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Manganese (Mn), an indispensable plant micronutrient, functions as a vital enzyme co-factor in numerous biochemical reactions. In rice, the Golgi-localized PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), a member of the UNCHARACTERIZED PROTEIN FAMILY (UPF0016), plays a pivotal role in Mn homeostasis, particularly in rapidly developing tissues. This study focused on the functional characterization of another UPF0016 family member in rice, OsPML4, to elucidate its involvement in Mn homeostasis. OsPML4 had a 73% sequence identity with OsPML3 and exhibited expression in both shoots and roots, albeit at a lower transcriptional level than OsPML3. Furthermore, subcellular localization studies confirmed that OsPML4 localizes in the Golgi apparatus. Notably, heterologous expression of OsPML4 restored growth in the Mn uptake-deficient yeast strain Δsmf1 under Mn-limited conditions. Under Mn-deficient conditions, OsPML4 knockout exacerbated the decline in shoot dry weight and intensified necrosis in young leaves of OsPML3 knockout lines, which displayed stunted growth. The Mn concentration in OsPML3PML4 double knockout lines was lower than in wild-type (WT) and OsPML3 knockout lines. At the reproductive phase, OsPML3PML4 double knockout lines exhibited reduced fertility and grain yield compared to WT and OsPML3 knockout lines. Notably, reductions were observed in the deposition of cell wall polysaccharides and the content of Le a (Lewis A structure)-containing N-glycans in the young leaves of OsPML3PML4 double knockout lines, surpassing the reductions in WT and OsPML3 knockout lines. These findings underscore the significance of OsPML4 in Mn homeostasis in the Golgi apparatus, where it co-functions with OsPML3 to regulate cell wall polysaccharide deposition and late-stage Golgi N-glycosylation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)