| Summary: | Broccoli (<i>Brassica oleracea</i> L. var. <i>italica</i>) is a globally popular vegetable because of its nutrient richness, especially its glucosinolates (GSLs). The content of GSLs in different organs of broccoli varies greatly. However, few studies have focused on the differences in the GSLs biosynthesis-related genes in different organs. In this study, we selected extreme individuals from an F<sub>2</sub> population of broccoli and mixed them to form low and high glucoraphanin content pools of leaf, stalk and floret, respectively. Transcriptome and metabolome analyses showed that 539, 755 and 617 genes are significantly differentially expressed, and 44, 66 and 118 metabolites are significantly differentially accumulated in leaf, stalk and floret comparison groups, respectively. The combined analysis revealed that some genes such as <i>Bo5g113720</i>, <i>Bo2g161100</i> and <i>Bo7g09800</i>, <i>Bo4g018590</i>, <i>Bo5g021810</i>, and <i>Bo2g011730</i> showed different expression trends between low and high glucoraphanin content pools, which increased the accumulation of glucoraphanin. These genes have different expression levels in the three plant parts. Strikingly, the accumulation of glucoraphanin upregulated the expression of plant hormone signal transduction-related genes <i>TIFY</i>, <i>JAR1</i>, <i>IAA</i>, <i>GH3</i> and <i>SAU</i>, and also increased the levels of tentatively identified flavonoid metabolites. Our study deepens the understanding of glucosinolate biosynthesis in different organs at the molecular level, and also provides evidence for the crosstalk between glucosinolates and flavonoids biosynthesis pathways.
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