Abstract
Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.
Highlights
Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress
Oxidation of ABAL and APAL is often attributed to the activity of NAD+-dependent aminoaldehyde dehydrogenases (AMADH, EC 1.2.1.19)[5,9], leading to GABA and β-alanine biosynthesis, respectively
Preliminary characterization indicated that initial rates of AtALADH10A8 and AtALDH10A9 activities display sharp pH optima at 10.5 and 9.5–9.7, respectively, at a saturating level of ABAL (Supplementary Fig. S2A) and subsaturating level of APAL (Supplementary Fig. S2B)
Summary
Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. The non-proteinogenic amino acid 4-aminobutyrate (GABA) accumulates in plants in response to various abiotic stresses such as chilling, drought and salinity[1,2] In dicotyledonous plants, it can be biosynthesized via two distinct pathways: from glutamate via pH- and calmodulin-dependent glutamate decarboxylase activity[3,4]; and, from terminal oxidation of the diamine putrescine and the polyamine spermidine via the action of copper-containing amine oxidase (CuAO, EC 1.4.3.22) and FAD-dependent polyamine oxidase (PAO, E.C. 1.5.3.6), respectively[5,6,7,8]. Oxidation of ABAL and APAL is often attributed to the activity of NAD+-dependent aminoaldehyde dehydrogenases (AMADH, EC 1.2.1.19)[5,9], leading to GABA and β-alanine biosynthesis, respectively. All kinetic curves are shown in Supplementary Figure S4. bVmax (μmol min−1 mg−1 protein), Km (μM), catalytic efficiency (kcat/Km, μM−1 s−1) and substrate inhibition constant (Kis, μM) are shown for each substrate or coenzyme
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