Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. In Gossypium hirsutum (cotton), four distinct trichomatic nectaries develop, one floral and three extrafloral. The secreted floral and extrafloral nectars serve different purposes, with the floral nectar attracting bees to promote pollination and the extrafloral nectar attracting predatory insects as a means of indirect resistance from herbivores. Cotton therefore provides an ideal system to contrast mechanisms of nectar production and nectar composition between floral and extrafloral nectaries. Here, we report the transcriptome, ultrastructure, and metabolite spatial distribution using mass spectrometric imaging of the four cotton nectary types throughout development. Additionally, the secreted nectar metabolomes were defined and were jointly composed of 197 analytes, 60 of which were identified. Integration of theses datasets support the coordination of merocrine-based and eccrine-based models of nectar synthesis. The nectary ultrastructure supports the merocrine-based model due to the abundance of rough endoplasmic reticulum positioned parallel to the cell walls and profusion of vesicles fusing to the plasma membranes. The eccrine-based model which consist of a progression from starch synthesis to starch degradation and to sucrose biosynthesis was supported by gene expression data. This demonstrates conservation of the eccrine-based model for the first time in both trichomatic and extrafloral nectaries. Lastly, nectary gene expression data provided evidence to support de novo synthesis of amino acids detected in the secreted nectars
