NTMC2T5 links lipid homeostasis to plastid differentiation.
Chloroplast biogenesis requires extensive lipid remodeling to establish the internal membrane systems of developing plastids, yet how lipid homeostasis is coordinated during this process remains incompletely understood. Here, we identify a previously unrecognized, Archaeplastida-conserved family of SMP-domain proteins and characterize its role in early plastid development. NTMC2T5 proteins contain an N-terminal chloroplast-targeting membrane region, an SMP domain, and a C2 domain, and localize in punctate patterns at the chloroplast envelope, enriched at regions associated with the endoplasmic reticulum (ER). Loss of NTMC2T5 in Nicotiana benthamiana causes severe defects in chloroplast development during seedling establishment and de-etiolation, whereas chloroplast maintenance in mature leaves is largely unaffected. Ultrastructural analyses revealed that mutant plastids fail to establish normal prolamellar bodies and organized thylakoid membranes, although plastid number and size were largely unaffected. Lipidomic analyses further revealed that NTMC2T5 loss causes a strong reduction in the plastid galactolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol, accompanied by accumulation of extraplastidial phospholipids and altered fatty-acid composition during de-etiolation. Together, these findings identify NTMC2T5 as a previously unrecognized determinant of lipid homeostasis during plastid differentiation and establish a link between a plant-specific SMP-domain protein family and chloroplast membrane biogenesis. We propose that NTMC2T5 contributes to ER-plastid lipid exchange and/or organization of ER-plastid membrane interfaces during early chloroplast development.