The surfaces of Bayberry (Myrica pensylvanica) fruits are covered with an extremely thick and unusual layer of crystalline wax. The surface wax accumulates to over 30% of the total fruit mass, the highest reported accumulation of surface lipids in nature. In addition, the composition of Bayberry wax is strikingly different from other plant surface waxes, consisting almost entirely of saturated triacylglycerol (TAG) and diacylglycerol (DAG), with palmitate and myristate as the dominant acyl chains. To gain insight into the unique properties of Bayberry surface wax production and secretion I have characterized the chemical and morphological development of the wax layer, the expression of gene transcripts in the wax secreting tissue and monitored wax biosynthesis through [14C]-acetate and [14C]-glycerol radiolabeling. Together these data provide evidence that Bayberry synthesizes TAG via a pathway not previously described in plants. Characterization of the accumulation of the wax layer identified monoacylglycerol (MAG) with its acyl-chain on the sn-2 position of glycerol (sn-2-MAG). sn-2 MAG was previously shown to be an early intermediate in the biosynthesis of the surface lipid polyesters cutin and suberin, and its accumulation in Bayberry wax suggested that Bayberry wax may be synthesized similarly. Indeed, in contrast to oilseed studies, radiolabeling identified sn-2 MAG as an initial labeled glycerolipid and the kinetics of [14C]-MAG labeling indicated a precursor-product relationship to DAG and TAG. Regiospecificity of the [14C]-acyl chains in DAG and TAG and their distribution between the wax and internal lipids strongly indicated that they were synthesized by transacylase activities with at least some reactions occurring extracellularly. RNA-seq of the wax producing tissue supported and extended the biochemical data. The most abundantly expressed transcripts during Bayberry wax accumulation were associated with surface lipid production, such as sn-2 glycerol-3-phosphate acyltransferases, GDSL lipases, ABC transporters, and LTPs. Transcripts encoding enzymes involved in seed TAG synthesis were 10-100 fold less abundant. The RNA seq data strongly support the hypothesis that a pathway related to cutin biosynthesis is responsible for the massive accumulation of extracellular TAG and DAG in Bayberry. This combination of a unique surface wax composition and massive secretion of lipids to the surface may help better understand how plants produce surface lipids, secrete them to the surface, and also to engineer alternative pathways to produce glycerolipids in non-seed tissue.
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