Edyta Zdunek-Zastocka

TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions

A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses (∼35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions

Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β-naphthylamides (β-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-β-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-β-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.

The activity pattern and gene expression profile of aldehyde oxidase during the development of Pisum sativum seeds

Aldehyde oxidase (AO, EC 1.2.3.1) is a molybdenohydroxylase that is considered to catalyze the final step in the synthesis of abscisic acid (ABA) and possibly of indole-3-acetic acid (IAA). Five AO activity bands were detected after native PAGE with indole-3-aldehyde (PsAO-α, -β, -γ, -δ, -κ) and three with abscisic aldehyde (PsAO-γ, -δ, -κ) in developing seeds of Pisum sativum. At early and mid-development, PsAO-α, -β, -γ and only PsAO-γ were observed, respectively, and their localization as well as the expression of PsAOs genes was almost exclusively restricted to the maternal fruit tissues, the seed coat and pericarp. Towards the end of rapid reserve synthesis, two additional isoforms (PsAO-δ, -κ) appeared in cotyledons, coinciding with a high transcript level of PsAO2. At this developmental stage, the activity level of PsAO-γ, was still considerable in the testa, and was higher than at earlier stages in the embryonic axis, which correlated with the PsAO3 transcript level. In mature dry seeds, AO activity and the expression of PsAOs became restricted to the embryonic tissues. The possible involvement of AO isoforms in ABA or IAA synthesis during pea seed development as well as the contribution of particular PsAO genes to the formation of the dimeric pea AO isoforms is discussed.

Plant molybdoenzymes and their response to stress

Molybdenum-containing enzymes catalyse basic reactions in the nitrogen, sulphur and carbon metabolism. Mo-enzymes contain at their catalytic sites an organometallic structure termed the molybdenum cofactor or Moco. In higher plants, Moco is incorporated into the apoproteins of four enzymes: nitrate reductase (EC 1.6.6.1-3; NR), xanthine dehydrogenase (EC 1.1.1.204; XDH), aldehyde oxidase (EC 1.2.3.1; AO) and sulphite oxidase (EC1.8.3.1; SO). Molybdoenzymes in plants are key enzymes in nitrate assimilation, purine metabolism, hormone biosynthesis, and most probably in sulphite detoxification. They are considered to be involved in stress acclimation processes and, therefore, elucidation of the mechanisms of their response to environmental stress conditions is of agricultural importance for the improvement of plant stress tolerance. Here we would like to give a brief functional and biochemical characteristic of the four plant molybdoenzymes and to focus mainly on their sensitivity to environmental stress factors.