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

Abstract

Summary The black nectar produced by Melianthus flowers is thought to serve as a visual attractant to bird pollinators, but the chemical identity and synthesis of the black pigment are unknown. A combination of analytical biochemistry, transcriptomics, proteomics, and enzyme assays was used to identify the pigment that gives Melianthus nectar its black color and how it is synthesized. Visual modeling of pollinators was also used to infer a potential function of the black coloration. High concentrations of ellagic acid and iron give the nectar its dark black color, which can be recapitulated through synthetic solutions containing only ellagic acid and iron(iii). The nectar also contains a peroxidase that oxidizes gallic acid to form ellagic acid. In vitro reactions containing the nectar peroxidase, gallic acid, hydrogen peroxide, and iron(iii) fully recreate the black color of the nectar. Visual modeling indicates that the black color is highly conspicuous to avian pollinators within the context of the flower. Melianthus nectar contains a natural analog of iron‐gall ink, which humans have used since at least medieval times. This pigment is derived from an ellagic acid‐Fe complex synthesized in the nectar and is likely involved in the attraction of passerine pollinators endemic to southern Africa.

A phosphatase gene is linked to nectar dihydroxyacetone accumulation in mānuka (Leptospermum scoparium)

“…However, although progress has been made on identifying the genetic basis for variation of nectar volume (Galliot et al, 2006;Wessinger et al, 2014;Barstow et al, 2022) or sucrose as a percentage of total sugars (Prasifka et al, 2018), such knowledge is limited for the other nectar components. Enzymes and/or candidate genes (Roy et al, 2022;Magner et al, 2023) and a QTL (Kostyun The accumulation of DHA in nectar of m anuka and some other species of Leptospermum results in honey, which is highly valued both as a food and as a medicinal product. The economic importance of m anuka honey has resulted in it being not only one of the only woody perennial species for which nectary biology and nectar traits have been studied in detail but also one of the few species with green, photosynthetic nectaries to have been characterised.…”

“…However, although progress has been made on identifying the genetic basis for variation of nectar volume (Galliot et al ., 2006; Wessinger et al ., 2014; Barstow et al ., 2022) or sucrose as a percentage of total sugars (Prasifka et al ., 2018), such knowledge is limited for the other nectar components. Enzymes and/or candidate genes (Roy et al ., 2022; Magner et al ., 2023) and a QTL (Kostyun et al ., 2019) have been identified responsible for nectar colour in a few species, but data on the control of production of other metabolites are lacking, perhaps because of the potential difficulties in obtaining sufficient phenotypic and genomic data. In this study, by growing mānuka plants from across Aotearoa‐NZ in a common environment and combining nectar screening with genetic sequence analysis of a population segregating for nectar DHA content, facilitated by a new genome sequence assembly, we were able to identify QTLs associated with this notable nectar trait.…”

“…Yet, studies on the molecular genetics of nectar production and secretion have focussed primarily on a few model species such as Arabidopsis thaliana (Arabidopsis; Kram & Carter, 2009), Nicotiana (Ren et al ., 2007; Silva et al ., 2018) and more recently squash ( Cucurbita pepo ; Solhaug et al ., 2019). Recently, genes or enzymes associated with the production of important specialised metabolites in nectar have also started to be characterised (Roy et al ., 2022; Magner et al ., 2023), but relatively few species have been studied in detail. Moreover, nectar composition can vary markedly even among species in a single genus.…”

Decoding the black essence of Melianthus nectar

“…All eight species of Melianthus have been reported to produce darkcolored nectar, ranging from light gray to black, while their most closely related genus Bersama produces clear nectar (Henning, 2003). The evolutionary history of these lineages makes This article is a Commentary on Magner et al (2023), 239: 2026-2040. them a great system to study the evolution of specialized nectar traits and the implication of these traits in pollinator interaction and reproductive success. Future studies investigating the genetic variation of the iron-ellagate complex production pathways in these closely related species will shed further light on the evolution of black nectar.…”

“…The latest to join the list of curious nectar compounds is the black pigment of Melianthus minor flowers. In an article published in this issue of New Phytologist, Magner et al (2023; 2026-2040 reported that ellagic acid, a heterotetracyclic compound that when complexed with ferric cations (Fe(III)), gives the nectar of M. minor flowers its characteristic black color. Coincidentally, this iron-ellagate complex is analogous to the iron-gallate and iron-tannate complexes that constitute the black pigment in iron-gall ink, the most common ink in use before the 20 th century (Fig.…”