Expression of Pisum sativum PsAO3 gene, which encodes an aldehyde oxidase utilizing abscisic aldehyde, is induced under progressively but not rapidly imposed drought stress

• Pisum sativum PsAO3 gene was heterologously expressed in Pichia pastoris.
• The recombinant PsAO3 revealed substrate preferences similar to the native PsAOγ.
• The PsAO3 revealed the lowest Km values for naphthaldehyde and abscisic aldehyde.
• Progressive but not rapid drought increased the level of PsAO3 mRNA and activity of PsAOγ.
• The concomitant involvement of AO and NCED in the stress-induced ABA synthesis is discussed.

Aldehyde oxidase (AO; EC 1.2.3.1) catalyzes the final step of abscisic acid (ABA) biosynthesis, which is the oxidation of abscisic aldehyde (ABAld) to ABA. Gene expression analyses indicate that the stress-induced Pisum sativum PsAOγ isoform, which effectively uses ABAld as a substrate, is encoded by the PsAO3 gene. PsAO3 was heterologously expressed in Pichia pastoris and the recombinant PsAO3 protein revealed substrate preferences highly similar to the native PsAOγ protein present in the pea leaves and roots. Both proteins prefer indole-3-aldehyde and naphthaldehyde as substrates, although high activities against abscisic aldehyde and citral were also observed. The Km values of PsAO3 for naphthaldehyde and abscisic aldehyde (4.6 and 5.1 μM, respectively) were the lowest among the substrates tested. PsAO3 activity was almost completely inhibited by potassium cyanide, diphenyleneiodonium, and methanol. Rapidly imposed drought stress did not increase the level of PsAO3 mRNA or activity of any AO isoform, although an enhanced ABA accumulation and induction of PsNCED2 and -3 (9-cis-epoxycarotenoid dioxygenase; EC 1.13.11.51) expression, both in the pea roots and leaves, was observed. During a progressively induced drought, the level of PsAO3 transcript and PsAOγ activity increased significantly in the roots and leaves, whereas ABA accumulation occurred only in the leaves where it was accompanied by induction of the PsNCED3 expression. Therefore, we suppose that next to NCED, also AO (mainly PsAOγ) might be involved in regulation of the drought-induced ABA synthesis. However, while the “constitutive activity” of PsAOγ is sufficient for the fast generation of ABA under rapid drought stress, the enhanced PsAOγ activity is required for the progressive and long-term ABA accumulation in the leaves under progressive drought stress.