ABSTRACT
Abscisic acid (ABA) plays a crucial role in plant defense mechanisms under adverse environmental conditions, but its metabolism and perception in response to heavy metals are largely unknown. In Pisum sativum exposed to CdCl2, an accumulation of free ABA was detected in leaves at different developmental stages (A, youngest, unexpanded; B1, youngest, fully expanded; B2, mature; C, old), with the highest content found in A and B1 leaves. In turn, the content of ABA conjugates, which was highest in B2 and C leaves under control conditions, increased only in A leaves and decreased in leaves of later developmental stages after Cd treatment. Based on the expression of PsNCED2, PsNCED3 (9-cis-epoxycarotenoid dioxygenase), PsAO3 (aldehyde oxidase) and PsABAUGT1 (ABA-UDP-glucosyltransferase), and the activity of PsAOγ, B2 and C leaves were found to be the main sites of Cd-induced de novo synthesis of ABA from carotenoids and ABA conjugation with glucose. In turn, ß-glucosidase activity and the expression of genes encoding ABA receptors (PsPYL2, PsPYL4, PsPYL8, PsPYL9) suggest that in A and B1 leaves, Cd-induced release of ABA from inactive ABA-glucosyl esters and enhanced ABA perception comes to the forefront when dealing with Cd toxicity. The distinct role of leaves at different developmental stages in defense against the harmful effects of Cd is discussed.
Subject(s)
Abscisic Acid , Cadmium , Gene Expression Regulation, Plant , Pisum sativum , Plant Leaves , Plant Proteins , Abscisic Acid/metabolism , Pisum sativum/metabolism , Pisum sativum/drug effects , Pisum sativum/genetics , Plant Leaves/metabolism , Plant Leaves/drug effects , Cadmium/metabolism , Cadmium/toxicity , Gene Expression Regulation, Plant/drug effects , Plant Proteins/metabolism , Plant Proteins/genetics , Dioxygenases/metabolism , Dioxygenases/genetics , beta-Glucosidase/metabolism , beta-Glucosidase/geneticsABSTRACT
The accumulation of proline is one of the defense mechanisms of plants against the harmful effects of adverse environmental conditions; however, when pea plants were treated for 12 h with CdCl2, the proline concentration decreased in the youngest A (not expanded) and B1 (expanded) leaves, and did not change significantly in the B2 (mature, expanded) or C (the oldest) leaves. After 24 h of cadmium (Cd) stress, the proline concentration remained low in A and B1 leaves, while in B2 and C leaves, it increased, and after 48 h, an increase in the proline concentration in the leaves at each stage of development was observed. The role of proline in the different phases of plant response to the Cd treatment is discussed. Changes in proline accumulation corresponded closely with changes in the transcript levels of PsP5CS2, a gene encoding D1-pyrroline-5-carboxylate synthetase involved in proline synthesis, and PsPDH1, a gene encoding proline dehydrogenase engaged in proline degradation. CdCl2 application induced the expression of PsProT1 and PsProT2, genes encoding proline transporters, especially during the first 12 h of treatment in A and B1 leaves. When the time courses of abscisic acid (ABA) and proline accumulation were compared, it was concluded that an increase in the proline concentration in the leaves of Cd-treated pea plants was more related to a decrease in chlorophyll concentration (leaves B2 and C) and an increase in the malondialdehyde level (A and B1 leaves) than with an increase in ABA concentration alone. Exogenous application of ABA (0.5, 5, 50 µM) significantly increased the proline concentration in the A leaves of pea plants only, and was accompanied by an elevated and repressed expression of PsP5CS2 and PsPDH1 in these leaves, respectively. The presented results suggest that under Cd stress, the accumulation of proline in leaves of pea plants may take place independently of the ABA signaling.
Subject(s)
Abscisic Acid/pharmacology , Cadmium/toxicity , Pisum sativum/metabolism , Pisum sativum/physiology , Plant Leaves/metabolism , Proline/metabolism , Stress, Physiological , Base Sequence , Biological Transport/genetics , Chlorophyll/metabolism , Gene Expression Regulation, Plant/drug effects , Genes, Plant , Malondialdehyde/metabolism , Pisum sativum/drug effects , Pisum sativum/genetics , Plant Leaves/drug effects , Proline/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stress, Physiological/drug effectsABSTRACT
Proline aminopeptidase (PAP, EC 3.4.11.5) is the only enzyme that effectively releases proline from the N-termini of peptides. The amino acid sequence of the PAP from Triticosecale, TsPAP1, comprises conserved regions, characteristic of the monomeric forms of PAP found in bacteria but not yet identified in plants. Therefore, we aimed to obtain and biochemically characterize the TsPAP1 protein. The recombinant TsPAP1 protein was received through heterologous expression of the TsPAP1 coding sequence in a bacterial expression system and purified with affinity chromatography. Gel filtration chromatography and SDS electrophoresis revealed that TsPAP1 is a monomer with a molecular mass of 37.5 kDa. TsPAP1 prefers substrates with proline at the N-terminus but is also capable of hydrolyzing ß-naphthylamides of hydroxyproline and alanine. Among the peptides tested, the most preferred were di- and tripeptides, especially those with glycine in the Y position. The use of diagnostic inhibitors indicated that TsPAP1 is a serine peptidase; however, further characterization revealed that the SH residues are also important for maintaining its activity. To examine the role of TsPAP1 under physiological conditions, we developed transgenic Arabidopsis plants overexpressing TsPAP1. Compared with wild-type plants, the transgenic lines accumulated more proline, flowered an average of 3.5 days earlier, and developed more siliques than did untransformed controls. Our paper is the first to describe the biochemical properties of a novel monomeric plant PAP and contributes to the functional characterization of PAP proteins in plants.
