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.

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, β-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.

The accumulation of proline, an important osmoprotective and antioxidant compound, is a key defense mechanism induced in plants in response to stress factors, including salinity, and is likely dependent on abscisic acid (ABA). However, in barley grown for 8 days under salinity conditions (125 mM NaCl), proline accumulation was not accompanied by changes in ABA content. Co-application of 0.5 mM molybdenum (Mo) significantly reduced NaCl-induced oxidative stress, as measured by H₂O₂, O₂⁻, MDA, and chlorophyll content, and increased the activity of Mo-containing aldehyde oxidase (AO), an enzyme involved in de novo ABA synthesis. As a result, elevated ABA levels were observed, but proline content under salinity conditions was similar in Mo-treated and non-Mo-treated plants. In contrast, exposing plants to 0.5 mM tungsten (W), an antagonist of Mo, inhibited AO activity without significantly altering ABA content, while proline and oxidative stress marker levels increased dramatically under both non-saline and saline conditions. The observed changes in proline content are mainly due to modulation of the rate of synthesis and, to a lesser extent, the rate of degradation, as revealed by transcript abundance of P5CS1 and PDH, which encode D1-pyrroline-5-carboxylate synthetase and proline dehydrogenase, respectively. The results indicate that in barley grown under salinity conditions, proline accumulation is ABA-independent but depends on the level of oxidative stress modulated by Mo and W ions.

Heavy metal pollution has become a major environmental challenge limiting agricultural productivity worldwide. Copper (Cu), although an essential micronutrient, becomes phytotoxic at elevated concentrations, primarily by inducing oxidative stress. Among the most widespread metabolic adjustments triggered by abiotic stress is the accumulation of proline, which is a compatible osmolyte that stabilizes proteins and membranes and helps maintain cellular redox homeostasis. This paper demonstrates that the heterologous expression of TsPAP1, a triticale gene encoding a prolyl aminopeptidase, enhances proline accumulation and confers increased Cu tolerance in Arabidopsis thaliana. Under Cu stress, transgenic lines maintained superior physiological performance relative to the wild type (WT), as evidenced by the reduced biomass loss and lower accumulation of malondialdehyde and reactive oxygen species. Cu exposure activated antioxidant defenses; however, the induction of catalase (CAT), class III peroxidases (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) activities was more pronounced in transgenic lines. Transcriptomic analysis revealed a higher expression of genes encoding antioxidant isoforms localized to chloroplasts (sAPX, CSD2), the cytosol (APX1), peroxisomes (CAT1), and the apoplast (Prx02, Prx51), indicating targeted reinforcement of the multi-compartmental redox defense system. Together, these findings identify TsPAP1 as a potential regulator of proline-dependent redox homeostasis that contributes to enhanced Cu tolerance through the coordinated activation of antioxidant networks. The results further suggest that PAP-dependent peptide turnover contributes to proline-mediated stress adaptation, linking peptide metabolism with antioxidant regulation and highlighting TsPAP1 as a promising target for engineering heavy metal-resilient crops.

Environmental contamination with heavy metals severely impairs crop productivity, primarily by inhibiting growth and triggering oxidative damage. To counteract these adverse effects, plants undergo complex metabolic reprogramming. Among these adaptations, the accumulation of purine-derived ureides—particularly allantoin—has been proposed as a protective mechanism, though its role under copper (Cu) stress remains unexplored. Here, we demonstrate that wild-type (Arabidopsis thaliana, WT) plants accumulate allantoate—the product of allantoin hydrolysis catalyzed by allantoinase (ALN, EC 3.5.2.5)—rather than allantoin when exposed to Cu toxicity. However, blocking this downstream catabolic step via loss of ALN function results in allantoin accumulation and is associated with enhanced Cu tolerance. Under Cu stress, aln-1 and aln-4 loss-of-function mutants maintained higher biomass and accumulated lower levels of malondialdehyde, superoxide anions, and hydrogen peroxide compared to WT. Furthermore, aln lines exhibited a stronger Cu-induced activation of key antioxidant enzymes (SOD, CAT, POD, APX) alongside higher expression of genes encoding specific isoforms targeted to multiple cellular compartments: cytosolic (CSD1), chloroplastic (CSD2, tAPX), peroxisomal (APX3, CAT3), and apoplastic (Prx51, Prx47). These results reveal that the loss of ALN function promotes a multi-compartmental antioxidant response under Cu toxicity, highlighting the pivotal role of purine catabolism in plant redox homeostasis and pointing to ureide metabolism as a potential avenue for enhancing crop stress tolerance.