Richard V. Espley

Anthocyanins play vital roles in plant physiology and offer human health benefits. Anthocyanin homeostasis is coordinately regulated by MYB activators and repressors. However, the molecular mechanisms behind the inhibitory roles of MYB repressors and their interplay with MYB activators remain to be fully determined. Here, we report that PpMYB308A, an R2R3-MYB repressor from peach, acts as an anthocyanin brake. PpMYB308A was capable of binding to DNA, and it competed with PpMYB10.1 for binding MBS cis-elements. PpMYB308A has an EAR motif at the C-terminus that could form a repressor complex with the co-repressors, TOPLESS protein PpTPL and histone deacetylase PpHDA6, to induce histone deacetylation at the promoter of target genes, thereby exerting active repressive function. PpMYB308A was able to bind the promoter of target genes, both directly by itself and indirectly by incorporating into the MYB-bHLH-WD10 (MBW) complex, to execute the EAR motif-mediated transcriptional repression. These results uncover an epigenetic mechanism behind the regulation of anthocyanin biosynthesis by R2R3-MYB repressors. Additionally, PpMYB10.1, the master regulator of anthocyanin biosynthesis in peach, could activate transcription of PpMYB308A, thereby forming a negative feedback loop to fine-tune anthocyanin accumulation.

Red fruit flesh is a rare occurrence in pears (Pyrus spp.), yet it is an attractive trait as anthocyanin enrichment would add nutritional value and novelty for consumers. Previous research has focused on red-skinned phenotypes in pear fruit, but the potential regulatory mechanisms controlling red flesh remain unclear. Here, we identified the Homeodomain leucine zipper (HD-ZIP) family transcription factor PyHAT5 as a controlling factor using transcriptome analysis of pear fruit flesh at 3 different developmental stages in a hybrid population of red-fleshed and white-fleshed pears. The expression level of PyHAT5 was significantly negatively correlated (correlation coefficient of −0.94) with anthocyanin content in the flesh. Overexpression of PyHAT5 inhibited anthocyanin accumulation in pear tissues (skin and callus), peach (Prunus persica) flesh, and tobacco (Nicotiana tabacum) leaves, while virus-induced silencing of PyHAT5 promoted intense coloration of the pear skin. Further analysis found that PyHAT5 protein interacts with PyMYB10, thereby blocking the formation and transcriptional activity of the PyMYB10–PybHLH3 complex. Additionally, we identified through yeast 2-hybrid screening that an E3 ubiquitin ligase, PyAIP2, interacts with PyHAT5 and may promote its ubiquitination and degradation. Overexpression of PyAIP2 promoted anthocyanin biosynthesis in the pear skin, while knockdown of PyAIP2 had the opposite effect. Our findings reveal a regulatory module, PyAIP2-PyHAT5-PyMYB10, that plays a critical role in regulating anthocyanin biosynthesis in red-fleshed pear. These findings also advance our understanding of the regulation of anthocyanin biosynthesis in other tissues and provide genetic knowledge for advancing breeding in pear.

Anthocyanins are important specialized fruit metabolites and major pigments, whose abundance depends on co-regulation of activators and repressors, primarily transcription factors (TFs) of the MYB family. Herein, a KANADI-like TF PuKAN4 was characterized in pear. This TF could be transcriptionally up-regulated by the anthocyanin-related R2R3-MYBs PuMYB10/PuMYB114 and exhibited high expression within red-skinned pears. Interestingly, PuKAN4 repressed anthocyanin biosynthesis in transiently overexpressed pear fruit, and stable transformation in pear calli and tobacco plants. The PuKAN4 had a conserved EAR repression domain in C-terminal, while the repression function of PuKAN4 could be offset by a transcription activation domain VP64. The dual luciferase analysis proved that PuMYB114/PuMYB10 up-regulated expression of PuKAN4. Furthermore, the PuKAN4 could physically interact with PuMYB10/PuMYB114 and did not affect the combination of MYB10/MYB114-bHLH3, as demonstrated by Y2H, pull-down and firefly luciferase complementation. Thus, the PuKAN4 should play the role of active repressor, the formation of PuKAN4-PuMYB10/PuMYB114-PubHLH3 complex inhibited pear anthocyanin biosynthesis. Our findings unveiled an activator-and-repressor feedback loop between PuMYB114/PuMYB10 and PuKAN4, which possibly balance biosynthesis activity and prevents over-accumulation of phenylpropanoids.

Blueberry is promoted as a super food with several health properties derived from chlorogenic acid and anthocyanin. Previous studies indicated that anthocyanin acylation and the content of chlorogenic acid could affect their level of absorption and biological activity. In this study, a genome-wide association study was performed to identify loci associated with anthocyanin and chlorogenic acid and characterize the candidate genes controlling anthocyanin acylation. Two stable loci controlling anthocyanin acylation and glucose specific glycosylation were confirmed on chromosomes 2 and 4, respectively, while no stable loci associated with chlorogenic acid were identified. Two acyl-CoA acyltransferases named VcBAHD-AT1 and VcBAHD-AT4 were identified as best candidate genes controlling anthocyanin acylation. Interestingly, the two genes clustered in acyl-CoA acyltransferases clade III, a clade that is not commonly associated with anthocyanin acylation. A virus-induced gene silencing approach optimized for silencing VcBAHD-AT1 and VcBAHD-AT4 in the whole blueberry fruits, confirmed the role of these two genes in anthocyanin acylation. Overall, this study establishes the foundation to develop a molecular marker to select for higher acylated anthocyanin and delivered a method for rapid functional characterization of genes associated with other fruit related traits in blueberry. Also, the study adds evidence that during the evolution of acyl-CoA acyltransferases multiple routes led to the emergence and/or fixation of the anthocyanin acyltransferase activity. These outcomes advance knowledge about the genes controlling anthocyanin acylation in blueberries and that extend to other plants. Selecting new blueberry cultivars with higher acylated anthocyanin levels could potentially increase absorption of this health-related bioactive.

Summary: The potential of cannabinoids to address public health challenges has stimulated exploration into alternative sources and production technologies. Radula marginata, an endemic Aotearoa/New Zealand liverwort, produces the bibenzyl cannabinoid perrottetinene (PET), analogous to Cannabis psychoactive tetrahydrocannabinol (THC). Structural differences between PET and THC could alter therapeutic interactions and mitigate adverse side effects. To understand the cannabinoid production potential of R. marginata, we analyzed 75 collections from three locations across several seasons, collaborating with kaitiaki Māori (indigenous guardians). Metabolic plasticity of the phytocannabinoids and plant growth was assessed under controlled growth conditions, and in in vitro culture. Perrottetinene diol (trans‐PTD), analogous to cannabidiol (trans‐CBD), and its acid precursor (PTDA), were identified and fully characterized from nature for the first time. Bibenzyl‐4‐geranyl (BB4G), analogous to cannabigerol (CBG), and its corresponding acid (BB4GA), were also isolated. Radula marginata showed chemotypes dominated by PET, PTD, or BB4G, in striking analogy to the main Cannabis chemotypes. These site‐selective chemotypes persisted after growth under artificial lighting and in in vitro progeny, suggesting genetic control. These results expand phytocannabinoid knowledge through the discovery of PTD analogous to CBD. They add a new dimension to liverwort cannabinoids and suggest convergent evolution of biosynthesis in two distant plant lineages.

Blueberry has a diversity of anthocyanins that confer its characteristic blue-coloured skin. Whilst most cultivars produce only anthocyanin glycosides, some can add aliphatic or aromatic groups to the sugar moiety to create acylated anthocyanins. Due to their enhanced stability, acylated anthocyanins represent an attractive breeding target in blueberry. In this study, a haplotype-resolved assembly of a previously identified quantitative trait locus on chromosome 2 of ‘Hortblue Petite’ (Vaccinium corymbosum) was created to identify candidate anthocyanin acyltransferase genes. One full-length gene (VcAAT1a) was selected based on quantitative PCR expression profiling and transient expression in tobacco leaves and in strawberry and blueberry fruit flesh. In all three systems, VcAAT1a was able to produce a range of acylated anthocyanins in planta. Recombinant VcAAT1a protein demonstrated that, while VcAAT1a was able to act on both anthocyanin 3-O-glucosides and 3-O-galactosides, it could only utilize acetyl-CoA as an acyl donor. Protein modelling using AlphaFold suggested that this restricted range in acyl donors may be due to a spatially restricted sub-pocket in the acyl-binding site of VvAAT1. Finally, LUC/REN promoter activation assays revealed that the VcAAT1a promoter was transactivated by the VcMYBPA1 and VcMYBPA2 transcription factors, further expanding our knowledge of anthocyanin regulation in blueberry.

Tea grey blight disease is one of the most destructive diseases that infects tea and is caused by the pathogen Pestalotiopsis theae (Sawada) Steyaert. L-theanine is a unique non-protein amino acid of the tea plant. Different concentrations of L-theanine exhibit significant inhibitory effects on the growth and sporulation ability of the pathogen causing tea grey blight disease. To understand the effect mechanism of L-theanine on P. theae, transcriptome profiling was performed on the pathogenic mycelium treated with three different concentrations of L-theanine: no L-theanine treatment (TH0), 20 mg/mL theanine treatment (TH2), and 40 mg/mL theanine treatment (TH4). The colony growths were significantly lower in the treatment with L-theanine than those without L-theanine. The strain cultured with a high concentration of L-theanine produced no spores or only a few spores. In total, 2344, 3263, and 1158 differentially expressed genes (DEGs) were detected by RNA-sequencing in the three comparisons, Th2 vs. Th0, Th4 vs. Th0, and Th4 vs. Th2, respectively. All DEGs were categorized into 24 distinct clusters. According to GO analysis, low concentrations of L-theanine primarily affected molecular functions, while high concentrations of L-theanine predominantly affected biological processes including external encapsulating structure organization, cell wall organization or biogenesis, and cellular amino acid metabolic process. Based on KEGG, the DEGs of Th2 vs. Th0 were primarily involved in pentose and glucuronate interconversions, histidine metabolism, and tryptophan metabolism. The DEGs of Th4 vs. Th0 were mainly involved in starch and sucrose metabolism, amino sugar, and nucleotide sugar metabolism. This study indicated that L-theanine has a significant impact on the growth and sporulation of the pathogen of tea grey blight disease and mainly affects amino acid metabolism, carbohydrate metabolism, and cellular structure-related biosynthesis processes of pathogenic fungi. This work provides insights into the direct control effect of L-theanine on pathogenic growth and also reveals the molecular mechanisms of inhibition of L-theanine to P. theae.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures), as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence–absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium—a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

For many fruit crops, the colour of the fruit outwardly defines its eating quality. Fruit pigments provide reproductive advantage for the plant as well as providing protection against unfavourable environmental conditions and pathogens. For consumers these colours are considered attractive and provide many of the dietary benefits derived from fruits. In the majority of species, the main pigments are either carotenoids and/or anthocyanins. They are produced in the fruit as part of the ripening process, orchestrated by phytohormones and an ensuing transcriptional cascade, culminating in pigment biosynthesis. Whilst this is a controlled developmental process, the production of pigments is also attuned to environmental conditions such as light quantity and quality, availability of water and ambient temperature. If these factors intensify to stress levels, fruit tissues respond by increasing (or ceasing) pigment production. In many cases, if the stress is not severe, this can have a positive outcome for fruit quality. Here, we focus on the principal environmental factors (light, temperature and water) that can influence fruit colour.