Physiological and proteomic responses of diploid and tetraploid black locust (Robinia pseudoacacia L.) subjected to salt stress

Abstract

Tetraploid black locust (Robinia pseudoacacia L.) is adaptable to salt stress. Here, we compared morphological, physiological, ultrastructural, and proteomic traits of leaves in tetraploid black locust and its diploid relatives under salt stress. The results showed that diploid (2×) plants suffered from greater negative effects than those of tetraploid (4×) plants. After salt treatment, plant growth was inhibited, photosynthesis was reduced, reactive oxygen species, malondialdehyde content, and relative electrolyte leakage increased, and defense-related enzyme activities decreased in 2× compared to those in 4×. In addition, salt stress resulted in distorted chloroplasts, swollen thylakoid membranes, accumulation of plastoglobules, and increased starch grains in 2× compared to those in 4×. However, 4× developed diverse responses under salt stress. A comparative proteomic analysis revealed that 41 and 37 proteins were differentially expressed in 2× and 4×, respectively. These proteins were mainly involved in photosynthesis, stress and defense, energy, metabolism, transcription/translation, and transportation. Distinct patterns of protein changes between 2× and 4× were analyzed. Collectively, our results suggest that the plants showed significantly different responses to salt stress based on ploidy level of the plant. The 4× possessed a better salt protection mechanism than that of 2×, suggesting salt tolerance in the polyploid plant.

Figure 1

Morphological changes in Robinia pseudoacacia diploid (2×) and tetraploid (4×) plants growth after salt treatment (500 mM NaCl). Diploid (A) and tetraploid (4×) (B) plants were grown in a soil and sand mixture (2:1) after 1, 5, 10, and 15 days of salt treatment.

Figure 2

Effects of salt stress on relative height growth rate (HGR) (A), relative range growth rate (RGR) (B), relative basal diameter growth rate (BGR) (C), and relative water content (RWC) (D) of leaves in Robinia pseudoacacia diploid (2×) (black bars) and tetraploid (4×) (white bars) plants growing under salt stress. Values followed by different letters are significantly different from each other at p < 0.05 according to Duncan’s method. Each data point represents the mean ± standard error of three replicates.

Figure 3

Effects of salt stress on O₂⁻ content (A), H₂O₂ content (B), and malondialdehyde (MDA) content (C) and relative electrolyte leakage (REL) (D) in the leaves of Robinia pseudoacacia diploid (2×) (black bars) and tetraploid (4×) (white bars) plants growing under salt stress. Values followed by different letters are significantly different from each other at p < 0.05 according to Duncan’s method. Each data point represents the mean ± standard error of three replicates.

Figure 4

Effects of salt stress on the concentrations of Na⁺ (A), Cl⁻ (B), K⁺ (C), and the K⁺/Na⁺ ratio (D) in the leaves of Robinia pseudoacacia diploid (2×) (black bars) and tetraploid (4×) (white bars) plants growing after salt treatment. Values followed by different letters are significantly different from each other at p < 0.05 according to Duncan’s method. Each data point represents the mean ± standard error of three replicates.

Figure 5

Effects of salt stress on net CO₂ assimilation rate (Pn) (A), stomatal conductance (Gs) (B), and intercellular CO₂ concentration (Ci) (C) in the leaves of Robinia pseudoacacia diploid (2×) (black bars) and tetraploid (4×) (white bars) plants growing under salt stress. Values followed by different letters are significantly different from each other at p < 0.05 according to Duncan’s method. Each data point represents the mean ± standard error of three replicates.

Figure 6

Effects of salt stress on the activities of superoxide dismutase (SOD) (A), peroxidase (POD) (B), ascorbate peroxidase (APX) (C), and glutathione reductase (GR) (D) in the leaves of Robinia pseudoacacia diploid (2×) (black bars) and tetraploid (4×) (white bars) plants growing after salt treatment. Values followed by different letters are significantly different from each other at p < 0.05 according to Duncan’s method. Each data point represents the mean ± standard error of three replicates.

Figure 7

Transmission electron micrographs of diploid (2×) and tetraploid (4×) Robinia pseudoacacia mesophyll cells after one day and 10 days of salt treatment. (A) 0 day, 2×; (B) 0 day, 4×; (C) 10 days, 2×; (D) 10 days, 4×. CW, cell wall; Ch, chloroplast; M, mitochondrion; Nu, nucleolus; P, plastoglobule; SG, starch granule; Gr, granum; V, vacuole; Vs, small vesicle.

Figure 8

Transmission electron microscopy observations of chloroplasts in diploid (2×) and tetraploid (4×) Robinia pseudoacacia after one day and 10 days of salt treatment. (A) 0 day, 2×; (B) 10 days, 2×; (C) 0 day, 4×; (D) 10 days, 4×. Gr, granum; thylakoid (Th); P, plastoglobule; SG, starch grain.

Figure 9

Coomassie Brilliant Blue (CBB)-stained two-dimensional electrophoresis gels of proteins from Robinia pseudoacacia diploid (2×) leaves. Proteins were separated on a 13 cm IPG strip (pH 4–7 linear gradient) using isoelectric focusing, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 12.5% gel. (A) 1-day NaCl-treated leaves; (B) 5-day NaCl-treated leaves; (C) 10-day NaCl-treated leaves. Blue and red numbers indicate proteins that increased and decreased between control and stressed samples, respectively. The proteins are listed in Table 1.

Figure 10

Coomassie Brilliant Blue (CBB)-stained two-dimensional electrophoresis gels of proteins from Robinia pseudoacacia tetraploid (4×) leaves. Proteins were separated on 13 cm IPG strip (pH 4–7 linear gradient) using isoelectric focusing, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 12.5% gel. (A) 1-day NaCl-treated leaves; (B) 5-day NaCl-treated leaves; (C) 10-day NaCl-treated leaves. Blue and red numbers indicate proteins that increased and decreased between control and stressed samples, respectively. The proteins are listed in Table 2.

Figure 11

Functional categorization of proteins in Robinia pseudoacacia diploid (2×) plants under salt stress. Digits indicate the protein number of each functional category.

Figure 12

Functional categorization of proteins in R. pseudoacacia tetraploid (4×) plants under salt stress. Digits indicate the protein number of each functional category.