Silencing of NtIPMDs is associated with impaired chloroplast development and altered amino acid metabolism in tobacco

Isopropylmalate dehydrogenase (IPMD) catalyzes the oxidation and decarboxylation of 3-isopropyl-L-malate (IPM) to form 4-methyl-2-oxovalerate in leucine biosynthesis. Although IPMD genes are well studied in Arabidopsis, their functions in other plants remain unclear. Here, we investigated two chloroplast-localized IPMDs (NtIPMD1 and NtIPMD2) in tobacco. RNAi-mediated silencing of both genes caused variegated leaves, stunted growth, reduced chlorophyll content, and abnormal chloroplast ultrastructure. Transcriptome analysis showed downregulation of photosynthesis-related genes, consistent with impaired photosynthetic capacity. Metabolomic profiling revealed that most amino acids, particularly valine, accumulated in NtIPMD -RNAi lines, whereas leucine increased only slightly. Phytohormone analysis further showed reduced ABA and GA levels. These results indicate that NtIPMDs silencing is associated with impaired chloroplast development and altered amino acid metabolism. The strong valine accumulation and slight leucine change suggest that leucine content may not directly reflect IPMD activity, and these effects may be indirect consequences of metabolic pathway. Further studies are needed to clarify whether NtIPMD has functions beyond leucine biosynthesis.