Ellagic acid/ellagitannins are plant polyphenolic antioxidants that are synthesized from gallic acid and have been associated with a reduced risk of cancer and cardiovascular diseases. Here, we report the identification and characterization of five glycosyltransferases (GTs) from two genera of the Rosaceae family (Fragaria and Rubus; F. × ananassa FaGT2*, FaGT2, FaGT5, F. vesca FvGT2, and R. idaeus RiGT2) that catalyze the formation of 1-O-galloyl-β-D-glucopyranose (β-glucogallin) the precursor of ellagitannin biosynthesis. The enzymes showed substrate promiscuity as they formed glucose esters of a variety of (hydroxyl)benzoic and (hydroxyl)cinnamic acids. Determination of kinetic values and site-directed mutagenesis revealed amino acids that affected substrate preference and catalytic activity. Green immature strawberry fruits were identified as the main source of gallic acid, β-glucogallin, and ellagic acid in accordance with the highest GT2 gene expression levels. Injection of isotopically labeled gallic acid into green fruits of stable transgenic antisense FaGT2 strawberry plants clearly confirmed the in planta function. Our results indicate that GT2 enzymes might contribute to the production of ellagic acid/ellagitannins in strawberry and raspberry, and are useful to develop strawberry fruit with additional health benefits and for the biotechnological production of bioactive polyphenols.

The CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) transcription factors (TFs) were repeatedly deployed over 725 million years of evolution to regulate central developmental innovations. The START domain of this pivotal class of developmental regulators was recognized over 20 years ago, but its putative ligands and functional contributions remain unknown. Here, we demonstrate that the START domain promotes HD-ZIPIII TF homodimerization and increases transcriptional potency. Effects on transcriptional output can be ported onto heterologous TFs, consistent with principles of evolution via domain capture. We also show the START domain binds several species of phospholipids, and that mutations in conserved residues perturbing ligand binding and/or its downstream conformational readout abolish HD-ZIPIII DNA-binding competence. Our data present a model in which the START domain potentiates transcriptional activity and uses ligand-induced conformational change to render HD-ZIPIII dimers competent to bind DNA. These findings resolve a long-standing mystery in plant development and highlight the flexible and diverse regulatory potential coded within this widely distributed evolutionary module.

Raspberries are becoming increasingly popular due to their reported health beneficial properties. Despite the presence of only trace amounts of anthocyanins, yellow varieties seems to show similar or better effects in comparison to conventional raspberries.

Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry ("Anne"). A 5 bp insertion in the coding region was identified and designated as ans+5. The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild-type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect, i.e., nonsense-mediated mRNA decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans+5 and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

Development of complex organisms requires the delicate and dynamic spatiotemporal regulation of gene expression. Central to this are microRNAs (miRNAs). These mobile small RNAs offer specificity in conveying positional information and versatility in patterning the outcomes of gene expression. However, the parameters that shape miRNA output during development are still to be clarified. Here, we address this question on a genome-wide scale, using the maize shoot apex as a model. We show that patterns and levels of miRNA accumulation are largely determined at the transcriptional level, but are finessed post-transcriptionally in a tissue-dependent manner. The stem cell environments of the shoot apical meristem and vasculature appear particularly liable to this. Tissue-specific effects are also apparent at the level of target repression, with target cleavage products in the vasculature exceeding those of other tissues. Our results argue against a clearance mode of regulation purely at the level of transcript cleavage, leading us to propose that transcript cleavage provides a baseline level of target repression, onto which miRNA-driven translational repression can act to toggle the mode of target regulation between clearance and rheostat. Our data show how the inherent complexities of miRNA pathways allow the accumulation and activity of these small RNAs to be tailored in space and time to bring about the gene expression versatility needed during development.