Massive Quantities of Glycerolipids in the Surface Wax of Bayberry (Myrica pensylvanica)

34 Bayberry (Myrica pensylvanica) fruits synthesize an extremely thick and unusual layer of 35 crystalline surface wax that accumulates to 32% of fruit dry weight, the highest reported surface 36 lipid accumulation in plants. The composition is also striking, consisting of completely saturated 37 triacylglycerol, diacylglycerol, and monoacylglycerol with palmitate and myristate acyl chains. 38 To gain insight into the unique properties of Bayberry wax synthesis we examined the chemical 39 and morphological development of the wax layer, monitored wax biosynthesis through [C]40 radiolabeling, and sequenced the transcriptome. Radiolabeling identified sn-2 MAG as an initial 41 glycerolipid intermediate. The kinetics of [C]-DAG and [C]-TAG accumulation and the 42 regiospecificity of their [C]-acyl chains indicated distinct pools of acyl donors and that final 43 TAG assembly occurs outside of cells. The most highly expressed lipid-related genes were 44 associated with production of cutin, whereas transcripts for conventional TAG synthesis were 45 >50-fold less abundant. The biochemical and expression data together indicate that Bayberry 46 surface glycerolipids are synthesized by a pathway for TAG synthesis that is related to cutin 47 biosynthesis. The combination of a unique surface wax and massive accumulation may aid 48 understanding of how plants produce and secrete non-membrane glycerolipids, and also how to 49 engineer alternative pathways for lipid production in non-seeds. 50