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Endosome trafficking has been reported to play an essential role in pollen tube polar growth and NtGNL1 (Nicotiana tabacum GNOM-LIKE 1) regulates the polar growth through endosome trafficking. However, the regulation network and detailed molecular mechanisms underlying endosome trafficking remain unclear. Here, comparative proteomic analysis was carried out to survey the overall effect of NtGNL1 on pollen tube polar growth and NtGNL1-dependent endosome trafficking. With multiple comparative systems (RNAi, Wild type, and BFA or wortmannin treatments), 481 distinct proteins were identified including 43 common DEPs (differentially expressed proteins), of which 16 significant DEPs were common among RNAi, BFA, and wortmannin treated pollen tubes, indicating their close relation to the endosome trafficking. GO annotation indicates that the vesicle trafficking of gnl1 HE pollen tubes differs from that of the BFA and wortmannin treated pollen tubes in the COPII-coated vesicle budding process. KEGG pathway analysis suggests that the Pentose phosphate pathway is critical for the NtGNL1-dependent endosome trafficking. Yeast two-hybrid further confirmed that the NtGNL1-Sec7 domain interacted strongly with VPS32.2, TCTP, PIS2, and PDIL2-1, suggesting that the core functional region of NtGNL1 is the Sec7 domain. Therefore, NtGNL1 likely functions via its Sec7 binding with these proteins to affect endosome trafficking. Our results provide a clear outline of proteins involving in NtGNL1-dependent endosome trafficking and valuable clues for understanding the regulatory mechanism of NtGNL1 guided pollen tube polar growth., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)