Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement

Mana Katano, Kazuki Takahashi, Tomonari Hirano, Yusuke Kazama, Tomoko Abe, Hirokazu Tsukaya, Ali Ferjani

Efficient use of seed nutrient reserves is crucial for germination and establishment of plant seedlings. Mobilizing seed oil reserves in Arabidopsis involves β-oxidation, the glyoxylate cycle, and gluconeogenesis, which provide essential energy and the carbon skeletons needed to sustain seedling growth until photoautotrophy is acquired. We demonstrated that H(+)-PPase activity is required for gluconeogenesis. Lack of H(+)-PPase in fugu5 mutants increases cytosolic pyrophosphate (PPi) levels, which partially reduces sucrose synthesis de novo and inhibits cell division. In contrast, post-mitotic cell expansion in cotyledons was unusually enhanced, a phenotype called compensation. Therefore, it appears that PPi inhibits several cellular functions, including cell cycling, to trigger compensated cell enlargement (CCE). Here, we mutagenized fugu5-1 seeds with (12)C(6+) heavy-ion irradiation and screened mutations that restrain CCE to gain insight into the genetic pathway(s) involved in CCE. We isolated A#3-1, in which cell size was severely reduced, but cell number remained similar to that of original fugu5-1. Moreover, cell number decreased in A#3-1 single mutant (A#3-1sm), similar to that of fugu5-1, but cell size was almost equal to that of the wild type. Surprisingly, A#3-1 mutation did not affect CCE in other compensation exhibiting mutant backgrounds, such as an3-4 and fugu2-1/fas1-6. Subsequent map-based cloning combined with genome sequencing and HRM curve analysis identified enoyl-CoA hydratase 2 (ECH2) as the causal gene of A#3-1. The above phenotypes were consistently observed in the ech2-1 allele and supplying sucrose restored the morphological and cellular phenotypes in fugu5-1, ech2-1, A#3-1sm, fugu5-1 ech2-1, and A#3-1; fugu5-1. Taken together, these results suggest that defects in either H(+)-PPase or ECH2 compromise cell proliferation due to defects in mobilizing seed storage lipids. In contrast, ECH2 alone likely promotes CCE during the post-mitotic cell expansion stage of cotyledon development, probably by converting indolebutyric acid to indole acetic acid.