Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase ( SAAT ) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli . The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short-and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

SummaryStrawberry (Fragaria 9 ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein).A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators.Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3Δ). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts.Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storagecompound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.Reproduction through seeds is a prominent feature of higher plants. Seeds are adapted to survive for periods of time under adverse conditions until conditions favorable for seedling establishment are encountered. Usually, mature seeds have low moisture contents, reduced metabolic activity, and have accumulated protective compounds to help them survive under rather severe conditions. In the course of seed maturation, various events happen including the accumulation of storage products, the suppression of precocious germination, the acquisition of desiccation tolerance, and often the induction of dormancy (for review, see Bewley and Black, 1994). Seeds become quiescent at desiccation and can often be stored for a long time. When nondormant dry seeds imbibe water, they can germinate to start a new lifecycle. During germination, a series of events occurs, such as the activation of respiration (Bewley and Black, 1994), the repair of macromolecules (Osborne, 1993), reserve mobilization (Gallardo et al., 2001), reinitiation of the cell cycle (De Castro et al., 1995; Vásquez-Ramos and Sánchez, 2004), and weakening of covering structures to allow radicle protrusion (Groot and Karssen, 1987). At the same time, seeds lose longevity during germination and desiccation tolerance upon radicle protrusion (Hong and Ellis, 1992). Our study focuses on these early events d...

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White spot syndrome virus, type species of the genus Whispovirus in the family Nimaviridae, is a large, double-stranded DNA (dsDNA) virus that infects crustaceans. The genome of the completely sequenced isolate WSSV-TH encodes 184 putative open reading frames (ORFs), the functions of which are largely unknown. To study the transcription of these ORFs, a DNA microarray was constructed, containing probes corresponding to nearly all putative WSSV-TH ORFs. Transcripts of 79 % of these ORFs could be detected in the gills of WSSV-infected shrimp (Penaeus monodon). Clustering of the transcription profiles of the individual genes during infection showed two major classes of genes: the first class reached maximal expression at 20 h post-infection (p.i.) (putative early) and the other class at 2 days p.i. (putative late). Nearly all major and minor structural virion-protein genes clustered in the latter group. These data provide evidence that, similar to other large, dsDNA viruses, the WSSV genes at large are expressed in a coordinated and cascaded fashion. Furthermore, the transcriptomes of the WSSV isolates WSSV-TH and TH-96-II, which have differential virulence, were compared at 2 days p.i. The TH-96-II genome encodes 10 ORFs that are not present in WSSV-TH, of which at least seven were expressed in P. monodon as well as in crayfish (Astacus leptodactylus), suggesting a functional but not essential role for these genes during infection. Expression levels of most other ORFs shared by both isolates were similar. Evaluation of transcription profiles by using a genome-wide approach provides a better understanding of WSSV transcription regulation and a new tool to study WSSV gene function.

To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin ochratoxin A (OTA). Variable domains (VHH) of heavy-chain antibodies binding to OTA were cloned from an immunised alpaca. Selected VHH clones clustered in four major groups, based on protein sequence similarity. Six representative VHH antibody fragments were produced in Escherichia coli and characterised for their sensitivity and specificity by indirect competitive enzyme-linked immunosorbent assays. OTA concentrations causing 50% inhibition (IC50) ranged from 12 ng/ml for antibody fragment OCH-62 to 442 ng/ml for antibody fragment OCH-40. The IC50 of OCH-62 for ochratoxin B, an OTA analogue, was 476 ng/ml. No significant cross-reactivity was observed with common food constituents with structural similarities with parts of OTA, such as L-phenylalanine and coumarin. The performance of OCH-62 for detection of OTA in food matrices was comparable to the performance in buffer (white wine: IC50=17 ng/ml; red wine: IC50=21 ng/ml; instant coffee: IC50=17 ng/ml). In a membrane-based flow-through immunoassay, used for fast visual screening of samples, OCH-62 showed a cut-off level of 10 ng/ml OTA. Thereby, OCH-62 ranked among the best recombinant antibody fragments described for mycotoxins and is an excellent candidate for the design of food contamination sensors based on nanotechnology.