EGY1 (ethylene-dependent gravitropism-deficient and yellow-green 1) is an intramembrane metalloprotease located in chloroplasts, involved in many diverse processes including chloroplast development, chlorophyll biosynthesis, and the ethylene-dependent gravitropic response. Plants deprived of this protease display pleiotropic effects such as the yellow-green early senescence phenotype and a poorly developed thylakoid system membrane in the mature chloroplasts. We applied the GC/MS technique to analyze the changes in fatty acid composition in two egy1 mutant lines. We used DAPI staining and transmission electron microscopy methods to establish the number of nucleoids and the amount of chloroplast DNA. Our results indicated that the lack of EGY1 protease led to a dramatic overaccumulation and a dramatic decrease in the content of linolenic acid C18:3 and hexadecatrienoic acid C16:3, respectively. The amount of chloroplast DNA and the number of nucleoids were severely reduced in egy1 mutant lines. Similarly, a reduced correlation between DAPI and autofluorescence signal was observed, which may indicate some perturbations in nucleoid anchoring.