Effect of Arsenic Soil Contamination on Stress Response Metabolites, 5-Methylcytosine Level and CDC25 Expression in Spinach

Abstract

Experimental spinach plants grown in soil with (5, 10 and 20 ppm) arsenic (As) contamination were sampled in 21 days after As(V) contamination. Levels of As in spinach samples (from 0.31 ± 0.06 µg g^-1 to 302.69 ± 11.83 µg g^-1) were higher in roots and lower in leaves, which indicates a low ability of spinach to translocate As into leaves. Species of arsenic, As(III) and As(V), were represented in favor of the As (III) specie in contaminated variants, suggesting enzymatic arsenate reduction. In relation to predominant As accumulation in roots, changes in malondialdehyde levels were observed mainly in roots, where they decreased significantly with growing As contamination (from 11.97 ± 0.54 µg g^-1 in control to 2.35 ± 0.43 µg g^-1 in 20 ppm As). Higher values in roots than in leaves were observed in the case of 5-methylcytosine (5-mC). Despite that, a change in 5-mC by As contamination was further deepened in leaves (from 0.20 to 14.10%). In roots of spinach, expression of the CDC25 gene increased by the highest As contamination compared to the control. In the case of total phenolic content, total flavonoid content, total phenolic acids content and total antioxidant capacity were higher levels in leaves in all values, unlike the roots.

Keywords: CDC25; arsenic species; epigenetics; membrane damage; secondary metabolites; spinach.

Figure 1

Content of phenolic compounds and total antioxidant capacity. TPC values in gallic acid equivalents (GAE) mg g⁻¹ DM, TFC values in quercetin equivalents (QE) mg g⁻¹ DM, PAC values in caffeic acid equivalents (CAE) mg g⁻¹ DM, TAC values in ascorbic acid equivalents (AAE) mg g⁻¹ DM. The letters indicate significant differences based on the post-hoc Tukey test, assuming p < 0.05.

Figure 2

Arsenic species ratio (%).

Figure 3

Results of the factorial ANOVA analysis for levels of 5-methylcytosine. The error bars indicate the standard deviations and the bar height is the mean value. Categories on the X-axis show four treatments (control, 5 ppm, 10 ppm and 20 ppm) values in roots and leaves. The letters indicate significant differences based on the post-hoc Tukey test, assuming p < 0.05.

Figure 4

Differences in the arrangement of the rhizodermis with a primary cortex, endodermis and vascular bundle with (100×) magnification.

Figure 5

Expression of CDC25 gene in spinach roots: (A); and leaves: (B) measured by qRT-PCR from 3 plants grown in all experimental variants. Values are mRNA average relative levels ± standard deviation of means in three biological replicates. The letters indicate significant differences based on the post-hoc Tukey test, assuming p < 0.05.

Figure 6

Relationships between the treatments and measured properties of the spinach evaluated using PCA (principal component analysis) with supplementary variables. The treatments are displayed using color circles and the arrows are measured variables or properties of the plants. The first two axes explain 91.45% of the total variation in the plant’s properties. RTL: relative transcript level.