Post-secretory synthesis of a natural analog of iron-gall ink in the black nectar of Melianthus spp.

The black nectar of Melianthus flowers is thought to serve as a visual attractant to pollinators, but the chemical identity and synthesis of the black pigment are unknown. Here we report that the black nectar contains a natural analog of iron-gall ink, which humans have used since medieval times. Specifically, dark black nectar at anthesis contains high levels of ellagic acid and iron; synthetic solutions of ellagic acid and iron(III) recapitulate the black color of the nectar. Conversely, lightly colored nectars before and after anthesis contain significantly lower levels of ellagic acid and iron, but higher levels of gallic acid. We then explored the possibility of post-secretory synthesis of ellagic acid from gallic acid. Indeed, Melianthus nectar contains a peroxidase that oxidizes gallic acid to form ellagic acid. Reactions containing the nectar peroxidase, gallic acid, hydrogen peroxide, and iron can fully recreate the black color of the nectar. Visual modeling indicates that the black color is both visible and conspicuous to birds within the context of the flower. In summary, the black nectar of Melianthus is derived from an ellagic acid-Fe complex analogous to iron-gall ink and is likely involved in the attraction of passerine bird pollinators. Overall design: Melianthus minor (also known as M. elongatus) plants were grown at the University of California Davis Arboretum and Public Garden at the University of California Davis campus in Davis, California, USA. Plants were grown and maintained outdoors on campus and received yearly maintenance of pruning and the application of fertilizer as needed. From 26 January 26 2022 to 1 February 2022, leaf, flower, nectary, and nectar samples were collected from the University of California Davis Arboretum and Public Garden. Collections took place each day between 10:00 AM and 5:00 PM. Previous studies have noted the terminal inflorescences of this species are sterile (Linder et al., 2006), but we noted they still produce some nectar. Plant tissue and nectar samples were collected and placed immediately in either a formalin aceto-alcohol (FAA) solution or an RNAlater stabilizing solution (Thermo Fisher Scientific catalog no. AM7020). Collected samples were stored at -80°C no later than February 4, 2022. Fresh leaf samples for fixation were collected and placed in 50 mL conical tubes containing FAA solution. Parafilm tape was used to seal the conical tubes, which were then stored at 4°C. Smaller leaf samples (~4 cm2) were collected in a 1.5 mL microcentrifuge tube containing an RNAlater stabilizing solution and immediately placed on dry ice before being stored at -80°C. Fresh flower samples were taken at various stages of development. Flowers were placed in FAA solution in 50 mL conical tubes. The conical tubes were wrapped in parafilm and stored at 4°C. Fresh inflorescences (Fig. S1) were collected and stored in 250 mL glass bottles with small amounts of sterile water and stored at 4°C until the collection of reflectance data. The collected nectary samples from fresh flowers occurred in the field and were placed in 1.5 mL microcentrifuge tubes with RNAlater stabilizing solution. The nectary samples (Fig. S1) were immediately placed in dry ice until stored at -80°C. The nectary dissections left some surrounding tissue intact, which was removed before further investigations occurred. Pipettes collected nectar in screw-top 1.5 mL microcentrifuge tubes and immediately placed on dry ice before storing at -80°C or were held at 4°C.