Spatial metabolome mass spectrometry imaging reveals the flavonoid distribution change in soybean nodules under drought and alkaline stresses

• Drought and alkali stress inhibited soybean growth and changed the metabolome of nodules. • The localization of isoflavone in nodules was altered under drought and alkaline stress. • The expression position of GmMaT2 is consistent with its metabolites and is regulated by GmbZIP59. Drought and alkaline stresses inhibit the symbiosis between soybean and rhizobia; however, the spatial distribution of metabolites within nodules under these stresses remains unknown. To address this gap, we used mass spectrometry imaging (MSI) to analyze metabolic alterations in different nodule regions under drought and alkaline stresses. A total of 3456 metabolites were detected in nodules and substances whose spatial distribution was altered by drought or alkaline stress were screened. Interestingly, the spatial distribution of the two most abundant isoflavones in soybean, malonyldaidzin and malonylgenistin, which serve as precursors for rhizobia-communication signaling molecules, was also affected by stresses. Spatial transcriptomic data revealed that the cortical-specific expression pattern of GmMaT2 (isoflavone malonyltransferase) matched the metabolite distribution. Further analysis identified GmbZIP59 as a transcriptional activator of GmMaT2, whose expression decreased under stress conditions. These results demonstrate that drought and alkaline stresses disrupt the spatial organization of flavonoid metabolism in nodules, potentially affecting symbiotic nitrogen fixation. Our findings provide new insights into the metabolic adaptation mechanisms of soybean nodules under abiotic stress.