Functional characterization of Arabidopsis thaliana isopropylmalate dehydrogenases reveals their important roles in gametophyte development

Abstract

Summary• Isopropylmalate dehydrogenases (IPMDHs) catalyze the oxidative decarboxylation of 3-isopropylmalate (3-IPM) in leucine biosynthesis in microorganisms. The Arabidopsis thaliana genome contains three putative IPMDH genes.• IPMDH2 and IPMDH3 proteins exhibited significantly higher activity toward 3-IPM than IPMDH1, which is indicative of a pivotal role in leucine biosynthesis. Single mutants of IPMDH2 or IPMDH3 lacked a discernible phenotype. Genetic analysis showed that ipmdh2 ipmdh3 was lethal in male gametophytes and had reduced transmission through female gametophytes. The aborted pollen grains were small, abnormal in cellular structure, and arrested in germination. In addition, half of the double mutant embryo sacs exhibited slowed development.• The IPMDH2 ⁄ ipmdh2 ipmdh3 ⁄ ipmdh3 genotype exhibited abnormal vegetative phenotypes, suggesting haplo-insufficiency of IPMDH2 in the ipmdh3 background. This mutant and a triple mutant containing one allele of IPMDH2 or IPMDH3 had decreased leucine biosynthetic enzyme activities and lower free leucine concentrations. The latter mutant showed changes in glucosinolate profiles different from those in the ipmdh1 mutant.• The results demonstrate that IPMDH2 and IPMDH3 primarily function in leucine biosynthesis, are essential for pollen development and are needed for proper embryo sac development.

Structural and Functional Evolution of Isopropylmalate Dehydrogenases in the Leucine and Glucosinolate Pathways of Arabidopsis thaliana

“…Bacterial expression vectors for each AtIPMDH (11,13) were used as templates with specific oligonucleotide pairs, as follows: AtIPMDH1-F137L-F, 5Ј-dCTGAGACCTGAGATGGCTCTGCTTTACC-TTAGAAGAGATCTC-3Ј; AtIPMDH1-F137L-R, 5Ј-dGAGA-TCTCTTCTAAGGTAAAGCAGAGCCATCTCAGGTCT-CAG-3Ј; AtIPMDH2-L133F-F, 5Ј-dCTGAGGCCTGAGAAG-GGGTTATTTCAGATTCGTGCAGCTCTC-3Ј; AtIPMDH2-L133F-R, 5Ј-dGAGAGCTGCACGAATCTGAAATAACCCC-TTCTCAGGCCTCAG-3Ј; AtIPMDH3-L134F-F, 5Ј-dCTG-AGACCTGAGATGGGTCTGTTTAACATTCGAAGAGA-3Ј; AtIPMDH3-L134F-R, 5Јd-TCTCTTCGAATGTTAAACA- GACCCATCTCAGGTCTCAG-3Ј (mutated codon in underlined and mutation shown in bold). Mutant protein expression, purification, and assays were performed as described above for wild-type enzyme.…”

“…AtIPMDH1 (At5g14200) is highly expressed in leaves and roots; AtIPMDH2 (At1g80560) is weakly expressed throughout the plant; and AtIPMDH3 (At1g31180) is constitutively expressed at high levels in all tissues (11,13,23). To test the possible contribution of differential expression to the specialization of AtIPMDHs, each gene was placed under control of the native AtIPMDH1 promoter and then transformed into an atipmdh1 mutant line (11).…”

“…Biochemical Analysis of Wild-type and Mutant AtIPMDHsPrevious studies on the AtIPMDHs demonstrate that each isoform accepts 3-isopropylmalate as a substrate (11,13), but a kinetic comparison with a glucosinolate pathway substrate has not been reported. Using both 3-isopropylmalate and 3-(2Ј-methylthio)ethylmalate, the steady-state kinetic parameters for each AtIPMDH were determined ( Table 2).…”

“…1B) (11,13). These studies highlight the functional specialization of these isoforms, but do not reveal how these activities evolved.…”

“…To date, all the genes involved in the methionine chain-elongation process have been identified and characterized in Arabidopsis thaliana (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The different enzymes of the methionine chain-elongation pathway for glucosinolate synthesis have evolved from leucine synthesis by gene duplication and functional specification (14 -15).…”