A hidden T-DNA-linked inversion-duplication is associated with a light-dependent Arabidopsis mutant phenotype

A hidden T-DNA-linked genomic rearrangement alters gene dosage and exacerbates light-dependent phenotypes in Arabidopsis, highlighting the importance of structural genome validation for reliable interpretation of mutantphenotypes. T-DNA insertion mutants are widely used to study gene function, yet associated genomic alterations can confound phenotypic interpretation. We investigated mutants affecting mitochondrial malate dehydrogenase (MDH1) and heterodimeric NAD-dependent malic enzyme (ME1/ME2) under different photoperiods and irradiance regimes. Under long-day conditions, mutant combinations showed no major phenotypic differences from wild type. Under short-day, low-light conditions, mdh1.1 me2 mutants were markedly smaller than wild type and, unexpectedly, more severely affected than mdh1.1 me1 me2 triple mutants. ME1 was undetectable in mdh1.1 me2, indicating that both genotypes effectively lack heterodimeric NAD-ME. Whole-genome analysis identified a 137-kbp inverted duplication downstream of the MDH1 insertion in mdh1.1 me2, absent from the triple mutant. The original mdh1.1 population segregated for the severe phenotype, whereas an independent mdh1.2 allele did not reproduce it, linking the defect to the mdh1.1-associated rearrangement. The duplication increased gene dosage and transcript abundance, particularly under low light. Proteomics detected only five of 38 proteins encoded within the duplicated region, including phosphoenolpyruvate carboxylase 1 (PEPC1). mdh1.1 me2 accumulated oxaloacetate-derived amino acids and exhibited altered carbon/nitrogen balance. These findings suggest that the duplication contributes to the phenotype, potentially through increased PEPC1 activity and consequent perturbation of carbon-nitrogen metabolism. However, interactions with MDH1 deficiency cannot be excluded, making PEPC1 a candidate contributor rather than the sole causal determinant. Our results highlight how T-DNA-linked structural variation can confound condition-dependent phenotypes and emphasize the need for structural validation of insertion loci, particularly given the widespread use of Agrobacterium-mediated DNA transfer in plant genome editing.