title: Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement.
abstract: 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.
authors.affiliation: Department of Biology, Tokyo Gakugei University Tokyo, Japan.
authors.forename: Mana
authors.initials: M
authors.lastname: Katano
authors.affiliation: Department of Biology, Tokyo Gakugei University Tokyo, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Biochemistry and Applied Biosciences, Miyazaki University Miyazaki, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Hirano
authors.affiliation: RIKEN Nishina Center Saitama, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kazama
authors.affiliation: RIKEN Nishina Center Saitama, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Abe
authors.affiliation: Department of Biological Sciences, Graduate School of Science, University of TokyoTokyo, Japan; Okazaki Institute for Integrative Bioscience, National Institutes of Natural SciencesOkazaki, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsukaya
authors.affiliation: Department of Biology, Tokyo Gakugei University Tokyo, Japan.
authors.forename: Ali
authors.initials: A
authors.lastname: Ferjani
keywords: Arabidopsis; Enoyl-CoA Hydratase 2; H+-PPase; compensation; cotyledons; gluconeogenesis; seed storage lipids