The structural and functional characterization of Malus domestica double bond reductase MdDBR provides insights towards the identification of its substrates.

In this study we describe the crystal structures of the apoform, the binary and the ternary complexes of a double bond reductase from Malus domestica L. (MdDBR) and explore a range of potential substrates. The overall fold of MdDBR is similar to that of the medium chain reductase/dehydrogenase/zinc-dependent alcohol dehydrogenase-like family. Structural comparison of MdDBR with Arabidopsis thaliana DBR (AtDBR), Nicotiana tabacum DBR (NtDBR) and Rubus idaeus DBR (RiDBR) allowed the identification of key amino acids involved in cofactor and ligands binding and shed light on how these residues may guide the orientation of the substrates. The enzyme kinetic for the substrate trans-4-phenylbuten-2-one has been analyzed, and MdDBR activity towards a variety of substrates was tested. This enzyme has been reported to be involved in the phenylpropanoid pathway where it would catalyze the NADPH-dependent reduction of the α, ß-unsaturated double bond of carbonyl metabolites. Our study provides new data towards the identification of MdDBR natural substrate and the biosynthetic pathway where it belongs. Furthermore, the originally proposed involvement in dihydrochalcone biosynthesis in apple must be questioned.

Broaden the sugar donor selectivity of blackberry glycosyltransferase UGT78H2 through residual substitutions.

Glycosylated secondary metabolites constitute a large proportion of nutrients or ingredients in consumed plants and related products. The glycosyl decoration largely depends on the activity of plant UDP-glycosyltransferases (UGTs). Mechanisms underlying the substrate selectivity and specificity of these reactions remain elusive. Here we report the cloning and functional characterization of a UGT, UGT78H2 in blackberry fruits. In vitro enzyme substrate specificity analysis and enzymatic kinetics evidenced that UGT78H2 glycosylate exclusively quercetin using uridine-5' diphosphate glucuronic acid (UDP-glucuronic acid) and uridine-5' diphosphate galactose (UDP-galactose). Site-directed mutagenesis was introduced into two residuals (N340P, K360N) previously unexplored. The mutation enhanced the protein catalyzing efficiency, especially toward UDP-galactose (23% higher), and expanded the sugar donor selectivity, which can use UDP-glucose as well. Molecular modeling and biochemical analysis results enable identification of the 23rd residue (360th in UGT78H2) of the PSPG (plant secondary product glycosyltransferase) motif as a key residue in defining this sugar selecting spectrum. Additionally, promoter of UGT78H2 was obtained. Transgenic analysis using the UGT78H2pro::GUS reporter system demonstrated that transcripts controlled by the promoter predominantly expressed in younger tissues. Subcellular localization study revealed that UGT78H2 was a soluble protein in the nucleus and cytoplasm. These results clarified the bio-function of UGT78H2 and provided a valid approach for substrate selectivity modification in horticultural plants, particularly for sugar donor selectivity.

Elicitation with Bacillus QV15 reveals a pivotal role of F3H on flavonoid metabolism improving adaptation to biotic stress in blackberry.

The aim of this study is to determine the involvement of the flavonol-anthocyanin pathway on plant adaptation to biotic stress using the B.amyloliquefaciens QV15 to trigger blackberry metabolism and identify target genes to improve plant fitness and fruit quality. To achieve this goal, field-grown blackberries were root-inoculated with QV15 along its growth cycle. At fruiting, a transcriptomic analysis by RNA-Seq was performed on leaves and fruits of treated and non-treated field-grown blackberries after a sustained mildew outbreak; expression of the regulating and core genes of the Flavonol-Anthocyanin pathway were analysed by qPCR and metabolomic profiles by UHPLC/ESI-qTOF-MS; plant protection was found to be up to 88%. Overexpression of step-controlling genes in leaves and fruits, associated to lower concentration of flavonols and anthocyanins in QV15-treated plants, together with a higher protection suggest a phytoanticipin role for flavonols in blackberry; kempferol-3-O-rutinoside concentration was strikingly high. Overexpression of RuF3H (Flavonol-3-hidroxylase) suggests a pivotal role in the coordination of committing steps in this pathway, controlling carbon flux towards the different sinks. Furthermore, this C demand is supported by an activation of the photosynthetic machinery, and boosted by a coordinated control of ROS into a sub-lethal range, and associated to enhanced protection to biotic stress.

Allopolyploid origin in Rubus (Rosaceae) inferred from nuclear granule-bound starch synthase I (GBSSI) sequences.

BACKGROUND: Polyploidy and hybridization are ubiquitous in Rubus L., a large and taxonomically challenging genus. Chinese Rubus are mainly concentrated into two major sections, the diploid Idaeobatus and the polyploid Malachobatus. However, it remains unclear to be auto- or allo- polyploid origin of polyploids in Rubus. We investigated the homoeologs and the structure of the GBSSI-1 (granule-bound starch synthase I) gene in 140 Rubus individuals representing 102 taxa in 17 (out of the total 24) subsections of 7 (total of 12) sections at different ploidy levels. RESULTS: Based on the gene structure and sequence divergence, we defined three gene variants, GBSSI-1a, GBSSI-1b, and GBSSI-1c. When compared with GBSSI-1a, both GBSSI-1b and GBSSI-1c have a shorter fourth intron, and GBSSI-1c had an additional deletion in the fifth intron. For diploids, either GBSSI-1a or GBSSI-1b was detected in 56 taxa consisting of 82 individuals from sect. Idaeobatus, while both alleles existed in R. pentagonus and R. peltatus. Both homoeologs GBSSI-1a and GBSSI-1b were identified in 39 taxa (48 individuals) of Malachobatus polyploids. They were also observed in two sect. Dalibardastrum taxa, in one sect. Chamaebatus taxon, and in three taxa from sect. Cylactis. Interestingly, all three homoeologs were observed in the three tetraploid taxa. Phylogenetic trees and networks suggested two clades (I and II), corresponding to GBSSI-1a, and GBSSI-1b/1c sequences, respectively. GBSSI-1 homoeologs from the same polyploid individual were resolved in different well-supported clades, and some of these homoelogs were more closely related to homoelogs in other species than they were to each other. This implied that the homoeologs of these polyploids were donated by different ancestral taxa, indicating their allopolyploid origin. Two kinds of diploids hybridized to form most allotetraploid species. The early-divergent diploid species with GBSSI-1a or -1b emerged before polyploid formation in the evolutionary history of Rubus. CONCLUSION: This study provided new insights into allopolyploid origin and evolution from diploid to polyploid within the genus Rubus at the molecular phylogenetic level, consistent with the taxonomic treatment by Yü et al. and Lu.

Enzymatic Profile of 'Willamette' Raspberry Leaf and Fruit Affected by Prohexadione-Ca and Young Canes Removal Treatments.

The influence of growth regulator prohexadione-Ca (ProCa) concurrently with young canes removal on the modification of photosynthetic pigments content and antioxidant enzymes (peroxidase, POD; catalase, CAT; polyphenol oxidase, PPO; superoxide dismutase, SOD) activities in leaves and fruits of raspberry (Rubus idaeus L.) cultivar 'Willamette' was studied. ProCa increased while canes removal decreased chlorophylls and carotenoids content compared to control. POD, CAT, and PPO activities in leaves after removal of young canes were higher compared to control (2-4 times) which was visually confirmed for POD by isoelectrofocusing. Removal of young canes slithly increased, while ProCa significantly enhanced SOD activity in leaves compared to control (475.10 and 218.38 nkat mg-1 prot, respectively). Pattern of SOD activity in fruit was similar as in leaf with substantial increase compared to control (about 15 times). Combination of implemented measures increased activity of all enzymes in the leaves and fruits. Our study could provide a better knowledge of the ProCa and canes removal influences on the action of enzymes in order to regulate their activities in fruit products.

Enzymatic inactivation and antioxidant properties of blackberry juice after thermoultrasound: Optimization using response surface methodology.

The purpose of this research was to optimize the thermoultrasound conditions for blackberry juice using the response surface methodology and considering juice quality parameters and antioxidant capacity. With the exception of microbial growth, the response variables showed high correlation coefficients with the mathematical model (R2adj>0.91). Thermoultrasound treatment inactivated all the evaluated microorganisms, and at the optimum conditions (50±1°C at 17±1min) it increased enzyme inactivation and antioxidant activity in comparison to pasteurized juice. The results demonstrated that thermoultrasound can be an alternative to pasteurization for the production of safe and high-quality juices with the added value of higher concentration of bioactive compounds and antioxidant capacity.

Differential expression of ethylene biosynthesis genes in drupelets and receptacle of raspberry (Rubus idaeus).

Red Raspberry (Rubus idaeus) is traditionally classified as non-climacteric, and the role of ethylene in fruit ripening is not clear. The available information indicates that the receptacle, a modified stem that supports the drupelets, is involved in ethylene production of ripe fruits. In this study, we report receptacle-related ethylene biosynthesis during the ripening of fruits of cv. Heritage. In addition, the expression pattern of ethylene biosynthesis transcripts was evaluated during the ripening process. The major transcript levels of 1-aminocyclopropane-1-carboxylic acid synthase (RiACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (RiACO1) were concomitant with ethylene production, increased total soluble solids (TSS) and decreased titratable acidity (TA) and fruit firmness. Moreover, ethylene biosynthesis and transcript levels of RiACS1 and RiACO1 were higher in the receptacle, sustaining the receptacle's role as a source of ethylene in regulating the ripening of raspberry.

Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production.

The flavour and fragrance compound ß-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for ß-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three ß-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of ß-carotene. To enable ß-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the ß-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, ß-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.