Unravel the molecular basis underlying inflorescence color variation in Macadamia based on widely targeted metabolomics

<i>Macadamia integrifolia</i>, a perennial evergreen crop valued for its nutritious nuts, also exhibits a diverse range of inflorescence colors that possess both ornamental and biological significance. Despite the economic importance of macadamia, the molecular mechanisms regulating flower coloration remain understudied. This study employed a combination of metabolomic and biochemical approaches to analyze metabolites present in inflorescences from 11 Macadamia cultivars, representing distinct color phenotypes. A total of 787 metabolites were identified through the use of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the majority of which were phenolic acids, flavonoids, and flavonols. Principal component analysis and clustering yielded a classification of the samples into three major flower color groups. The differential metabolites were found to be enriched in pathways such as flavonoid, flavonol, and phenylpropanoid biosynthesis, which have been demonstrated to be key contributors to color variation. Moreover, weighted gene co-expression network analysis (WGCNA) identified metabolite modules that were strongly associated with specific flower colors. This revealed that key compounds, including kaempferol, quercetin derivatives, and anthocyanins, were the primary drivers of pigmentation. This study provides a comprehensive framework for understanding the genetic, biochemical, and environmental factors influencing macadamia flower color. These findings contribute to the theoretical understanding of macadamia reproductive biology and have practical implications for molecular breeding, ornamental enhancement, and optimizing pollinator attraction to improve crop yield and ecological sustainability.