Investigation of the chromium(VI) tolerance and the roles of chromium(VI) and sulfate on the proteome changes in canola (Brassica napus L.) seedlings

In the first stage of this study, the phytoremediation potential of 8 canola (Brassica napus L.) cultivars (Champlain, Dante, Heros, Nelson, NK Petrol, Sary, Süzer and Vectra) for hexavalent chromium [Cr(VI)] were evaluated in hydroponic culture. For this purpose, the alterations in seedling growth, Cr accumulation, malondialdehyde (MDA) content, chlorophyll content, non-protein thiol content (NPT) and activities of some antioxidant enzymes [superoxide dismutase (SOD), guaiacol peroxidase (POD) and catalase (CAT)] were investigated in canola seedlings exposed to Cr(VI) stress (0, 10, 50 and 100 µM potassium dichromate) for 7 days. Chromium stress caused a reduction in dry weights (DW), chlorophyll content and CAT activity, and increase in lipid peroxidation, NPT content and activities of SOD and POD. Based on the dry weight, the tolerance index was found maximum in cultivar NK Petrol (%82.5-74.1) and minimum in cultivar Sary (%65.7-40.8). The cultivar Sary accumulated the maximum amount of Cr (705.8 μg g1 DW), which was correlated with the lowest levels of chlorophyll content and highest levels of lipid peroxidation. However, Cr accumulation was found lowest (255.0 μg g1 DW) in NK Petrol. Moreover, the cultivar NK Petrol maintained high content of chlorohyll and CAT activity under Cr(VI) stress. Although NK Petrol may be a Cr(VI) excluder cultivar relative to Sary, it may have the potential for the phytoremediation of Cr-contaminated sites as it tolerates Cr(VI) stress by producing higher biomasses. In the second stage of this study, the interactions between sulfur (S) nutrition and Cr(VI) stress were evaluated in relatively Cr-tolerant (NK Petrol) and Cr-sensitive (Sary) canola cultivars differing in Cr(VI) tolerance. The canola seedlings were grown S-sufficient (1 mM S) nutrient solution for 7 days. The seedlings were then grown in variable S concentrations [0 mM S (–S), 1 mM S (+S) and 2 mM S (+HS)] for another 7 days. The fourteen-day-old seddlings were exposed to 6 different treatments for 3 days: (1) +S/−Cr; 1 mM sulfate, 0 μM Cr(VI) (control), (2) +S/+Cr; 1 mM sulfate, 100 μM Cr(VI), (3) −S/−Cr; 0 mM sulfate, 0 μM Cr(VI) (sulfur deficiency), (4) −S/+Cr; 0 mM sulfate, 100 μM Cr(VI) (combined stress), (5) +HS/−Cr; 2 mM sulfate, 0 μM Cr(VI) (excess S treatment) and (6) +HS/+Cr; 2 mM sulfate, 100 μM Cr(VI). After this period, the seedlings were used to evaluate the alterations in growth, Cr accumulation, lipid peroxidation, chlorophyll content, thiol content, cysteine content, ascorbic acid (AsA) and glutathione (GSH) contents, activities of some antioxidant enzymes [SOD, POD, CAT, ascorbate peroxidase (APX), glutathione S-transferase (GST) and glutathione reductase (GR)], expression level of metallothionein protein 1 gene (BnMP1) and leaf proteome. The –S/–Cr and –S/+Cr treatments caused a marked decrease in growth parameters of cultivar Sary. The maximum Cr accumulation was measured in –S/+Cr plants, especially in cultivar Sary. The higher level of Cr in cultivar Sary increased lipid peroxidation and decreased chlorophyll content. The activities of antioxidant enzymes and cysteine content were found higher in NK Petrol under –S/+Cr condition. The levels of antioxidant metabolites (AsA and GSH) in both cultivars were decreased by –S/–Cr treatment. The expression level of BnMP1 gene was increased by –S/+Cr treatment in NK Petrol, while incrased +S/+Cr and +HS/+Cr treatments in Sary. This result suggests metallothionein protein may be involved in Cr(VI) tolerance but only under S-deficiency not during availability of S. In conclusion, the tolerace was increased by the positive effects of sulfur nutrition on defense systems and gene expression as well as by the preventing Cr uptake. In the present study, the proteome changes related to sulfur nutrition and Cr(VI) stress were determined in a relatively Cr-tolerant NK Petrol and a Cr-sensitive Sary cultivars of canola. Leaf protein profiles analyzed by two-dimensional gel electrophoresis revealed that 65 protein spots were differentially expressed in response to variable S concentrations and Cr(VI) stress. Of these, differentially expressed 46 protein spots were identified successfully by MALDI-TOF/TOF mass spectrometry and database search. Majority of the differentially expressed proteins were involved in photosynthesis, energy metabolism and stress defense. In relation to stress defense, 2-Cys peroxiredoxin BAS1, glutathione S-transferase, ferritin-1, L-ascorbate peroxidase 1, thiazole biosynthetic enzyme and myrosinase-binding protein-like exhibited a greater increase in response to sulfur deficiency and Cr(VI) stress in the relatively Cr-tolerant NK Petrol compared to Cr-sensitive Sary. It is suggested that the greater increase in the abundance of proteins involved in energy metabolism in cultivar NK Petrol might help to Cr(VI) stressed cells to meet the high energy demand. On the other hand, it is suggested that the enhanced expression of molecular chaperone (20 kDa chaperonin) in NK Petrol might be another additional defense mechanism for refolding of misfolded proteins and to stabilize protein structure and function. The findings of the present proteomics study provide the primary information regarding the molecular basis of sulfur deficiency and Cr(VI) tolerance in canola plants.