A T-DNA-induced chromosomal translocation in smo2-2 disrupts gametophyte development independently of sterol biosynthesis

Abstract The sterol C-4 demethylase multienzyme complex (SC4DM), containing the sterol 4α-methyl oxidases SMO1 and SMO2, plays essential roles in plant reproduction. In Arabidopsis (Arabidopsis thaliana), 2 SMO2 genes (SMO2-1 and SMO2-2) encode functionally redundant enzymes, as evidenced by the embryo-lethal phenotype and auxin-related patterning defects observed in smo2-1 smo2-2 double mutants. Intriguingly, while smo2-1/+ smo2-2 plants develop normally, smo2-1 smo2-2/+ heterozygotes produce siliques with ∼50% unfertilized ovules, suggesting a genotype-dependent effect whose mechanism requires elucidation. Here, we found that the SMO2 genes are highly expressed during anther and ovule development, with SMO2-1 showing stronger expression than SMO2-2. The smo2-1 smo2-2/+ mutant exhibits ∼50% abortion rates for both male and female gametophytes, with developmental arrest occurring around the first mitosis. Through comprehensive analysis, we excluded several potential mechanisms for these aberrant phenotypes, including auxin deficiency, SMO2-2 splice variants, and secondary T-DNA insertions. Instead, we identified a reciprocal chromosomal translocation induced by T-DNA insertions in smo2-2 as the underlying cause. This conclusion was further supported by characterizing a knockdown allele (smo2-1 smo2-2a/+) and a CRISPR/Cas9 knockout line (smo2-1 smo2-2k1/+), which confirmed that the observed gametophytic defects are independent of SMO2 loss-of-function. These findings highlight the necessity of analyzing multiple independent alleles when interpreting mutant phenotypes.