Proline Concentration and Its Metabolism Are Regulated in a Leaf Age Dependent Manner But Not by Abscisic Acid in Pea Plants Exposed to Cadmium Stress

Abstract

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.

Keywords: Pisum sativum L.; abscisic acid; cadmium; heavy metals; malondialdehyde; proline dehydrogenase; proline transporters; pyrroline-5-carboxylate synthetase.

Figure 1

Changes in the chlorophyll (A) and malondialdehyde (B) concentration in the leaves of pea plants exposed to cadmium (Cd) treatment. Cadmium was applied as 50 µM CdCl₂. A, the youngest unexpanded leaves; B1, the youngest fully expanded leaves; B2, fully expanded mature leaves; and (C), the oldest true leaves. The results are the means (±SD) of three biological replicates. Significant differences (at least p < 0.05) between the means are shown above the columns by different letters: lowercase between different time points in a group of leaves with the same stage of development, and capital between leaves with different stages of development but at the same time point.

Figure 2

Changes in the proline concentration (A) and transcript level of PsP5CS1 (B), PsP5CS2 (C), and PsPDH1 (D) after 12, 24, and 48 h of Cd treatment. Cadmium was applied as 50 µM CdCl₂. The relative mRNA level in individual leaves was expressed in relation to that in A leaves of control plants, set to 100, after normalization to reference genes. A, the youngest unexpanded leaves; B1, the youngest fully expanded leaves; B2, fully expanded mature leaves; and C, the oldest true leaves. The results are the means (±SD) of three biological replicates. Significant differences (at least p < 0.05) between the means are shown above the columns by different letters: lowercase between different time points in a group of leaves with the same stage of development, and capital between leaves with different stages of development but at the same time point.

Figure 3

Changes in the transcript level of PsProT1 (A) and PsProT2 (B) after 12, 24, and 48 h of Cd treatment. Cadmium was applied as 50 µM CdCl₂. The relative mRNA level in individual leaves was expressed in relation to that in A leaves of control plants, set to 100, after normalization to reference genes. A, the youngest unexpanded leaves; B1, the youngest fully expanded leaves; B2, fully expanded mature leaves; and C, the oldest true leaves. The results are the means (±SD) of three biological replicates. Significant differences (at least p < 0.05) between the means are shown above the columns by different letters: lowercase between different time points in a group of leaves with the same stage of development, and capital between leaves with different stages of development but at the same time point.

Figure 4

Changes in the abscisic acid concentration after 12 h of Cd treatment. Cadmium was applied as 50 µM CdCl_2. A, the youngest unexpanded leaves; B1, the youngest fully expanded leaves; B2, fully expanded mature leaves; and C, the oldest true leaves. The results are the means (±SD) of three biological replicates. Different letters above the columns indicate significant differences between the means (p < 0.05).

Figure 5

Changes in the proline concentration (A), abscisic acid (ABA) concentration (B), and transcript levels of PsP5CS2 (C) and PsPDH1 (D) after 24 h of ABA treatment. ABA was applied at a concentration of 50 µM. The relative mRNA level in individual leaves was expressed in relation to that in A leaves of control plants, set to 100, after normalization to reference genes. A, the youngest unexpanded leaves; B1, the youngest fully expanded leaves; B2, fully expanded mature leaves; and C, the oldest true leaves. The results are the means (±SD) of three biological replicates. Different letters above the columns indicate significant differences between the means (p < 0.05).

Figure 6

Changes in the transcript levels of PsProT1 (A) and PsProT2 (B) after 24 h of ABA treatment. ABA was applied at a concentration of 50 µM. The relative mRNA level in individual leaves was expressed in relation to that in A leaves of control plants, set to 100, after normalization to reference genes. A, the youngest unexpanded leaves; B1, the youngest fully expanded leaves; B2, fully expanded mature leaves; and C, the oldest true leaves. The results are the means (±SD) of three biological replicates. Different letters above the columns indicate significant differences between the means (p < 0.05).