Root and floral effects of flavonoid transport via an Arabidopsis MATE family transporter

Amongst their numerous roles in plants, flavonoid pigments are hypothesised to affect growth and development via interactions with components of auxin transport networks. Accordingly, our previous work showed that a multidrug and toxin efflux family transporter (FFT) alters root and seed characteristics and was required for full fertility in Arabidopsis. FFT is transcribed in guard cells throughout the plant and inactivation of FFT caused a significant perturbation in flavonoid profile in floral organs. Indeed, SEM and viability staining of mutant flowers reveal reduced anther dehiscence and a proportion of defective pollen, while siliques are smaller with fewer seeds than in WT. Null mutant seedlings grow faster and bolt sooner than WT, and seed size and mucilage are also affected. We are currently quantifying in more detail the flavonoid content of fft plants, and investigating the effect of externally applied auxin, flavonols and an auxin transport inhibitor. Finally, since we see FFT- promoter-GUS induced staining in vegetative tissues, including hydathode guard cells, we are also examining the abiotic stress response in the fft mutant. Co-expressed genes are involved in drought response including salt and osmotic stress

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date

A method for visualising fluorescence of flavonoid therapeutics in vivo in the model eukaryote Dictyostelium discoideum

Naturstoff reagent A (diphenylboric acid 2-aminoethyl ester, DPBA) has been used historically in plant science to observe polyphenolic pigments, such as flavonoids, whose fluorescence requires enhancement to be visible by microscopy. Flavonoids are common dietary constituents and are the focus of considerable attention because of their potential as novel therapies for numerous diseases. The molecular basis of therapeutic activity is only gradually being established, and one strand of such research is making use of the social amoeba Dictyostelium discoideum. We extended the application of DPBA to flavonoid imaging in these preclinical studies and report the first method for use of DPBA in this eukaryotic model microbe, and its applicability alongside subcellular markers. This in vivo fluorescence imaging provided a useful adjunct to parallel chemical and genetic studies

Development of flavonoid therapeutics: Transport mechanisms in the model eukaryote Dictyostelium discoideum

We are investigating the function of two members of the multidrug and toxin efflux (MATE) transporter family, which are ubiquitous throughout all living kingdoms. MATEs have a wide range of substrate targets, with transporters conferring the ability to facilitate the movement of single or many compounds (Omote et al., 2006). We have identified two genes encoding 'MATE' proteins in the model amoeba Dictyostelium discoideum, and are making knockout and reporter lines for physiological and imaging studies. Expression of the transporters at different life stages suggests the two have distinct and not redundant functions. The first transporter gene is transcribed in highest levels when cells signal to each other and aggregate. The second transporter gene is predominant earlier, when unicellular Dictyostelium cells prey on bacteria, and transcription also peaks later when cells have aggregated to form a motile, multicellular slug. We hope to address fundamental questions in the cell biology of this important model organism such as the role of the extracellular matrix and how it is formed; to study localisation of these proteins in the unicellular and multicellular life cycle; and also to contribute to work on the mechanisms of a flavonoid therapy for polycystic kidney disease which is under development by our collaborators (Waheed et al., 2014). In plants, it is hypothesised that so-called 'MATE' transporter proteins may be involved in flavonoid transport: currently there is sparse literature on the transport mechanisms that would allow/prevent this family of compounds reaching eukaryotic cellular targets