Beyond defense: GTR-mediated uptake of glucosinolates drives growth and structural reprogramming in broccoli seedlings.

Exogenous application of glucosinolates (GSLs) has previously been reported to act as a biostimulant, enhancing crop resilience and growth. However, the molecular mechanisms governing their uptake and metabolic integration remain unknown. In this study, we investigated the role of glucosinolate transporters (GTRs) in the active internalization of exogenous GSLs and their subsequent impact on the early development of broccoli (Brassica oleracea L. var. italica) seedlings. Using a multidisciplinary approach combining Xenopus laevis oocyte heterologous expression, confocal microscopy with fluorescent GSL derivatives, and RNA-seq analysis, we demonstrated that broccoli seedlings actively uptake exogenous GSLs through the root system. Accordingly, functional characterization confirmed that BolGTR1.2, BolGTR2.2, BolGTR2.3, and BolGTR3.3 are high-efficiency transporters capable of translocating both aliphatic and indolic GSLs. Together, GSL profiling, time-course RT-qPCR, and Day 7 RNA-seq revealed that GSL internalization triggers a biphasic response: an initial "consumption phase," followed by a compensatory upregulation of de novo GSL biosynthetic machinery by Day 7. This metabolic "shortcut" bypasses the energetically costly sulfate reduction pathway, allowing for the reallocation of cellular energy toward structural growth, which was evidenced by the induction of cell wall remodeling genes (XTH, PAE8, GAE) and a measurable increase in seedling biomass. Collectively, our findings support the hypothesis that GSLs are not merely defensive compounds but dynamic metabolic reserves that coordinate resource allocation.