Molecular, Biochemical and Ultrastructural Changes Induced by Pb Toxicity in Seedlings of Theobroma cacao L.

Pb is a metal which is highly toxic to plants and animals, including humans. High concentrations of Pb have been observed in beans of T. cacao, as well as in its products. In this work, we evaluated the molecular, biochemical, and ultrastructural alterations in mature leaves and primary roots of seedlings of two progenies of T. cacao, obtained from seed germination in different concentrations of Pb (0, 0.05, 0.1, 0.2, 0.4, 0.8 g L(-1)), in the form of Pb(NO3)2. The progenies resulted from self-fertilization of Catongo and a cross of CCN-10 x SCA-6. The Pb, supplied via seminal, caused alterations in the ultrastructures of the mesophyll cells and in the amount of starch grains in the chloroplasts. The dosage of substances reactive to thiobarbituric acid showed that Pb induced lipid peroxidation. The activity of guaiacol peroxidases and the expression of genes associated to synthetase of phytochelatin, SODcyt and PER increased in response to Pb. In addition, there was alteration in the expression of stress-related proteins. The progeny of CCN-10 x SCA-6 was more tolerant to Pb stress when compared to Catongo, since: (i) it accumulated more Pb in the roots, preventing its translocation to the shoot; (ii) it presented higher activity of peroxidases in the roots, which are enzymes involved in the elimination of excess of reactive oxygen species; and (iii) increased expression of the gene in the phytochelatin biosynthesis route. The results of the proteomic analysis were of paramount importance to differentiate the defense mechanisms used by both progenies of T. cacao.

Accumulation, detoxification, and genotoxicity of heavy metals in Indian mustard (Brassica juncea L.).

Plants of Indian mustard (Brassica juncea L.) were exposed to different concentrations (15, 30, 60, 120 microM) of (Cd, Cr, Cu, Pb) for 28 and 56 d for accumulation and detoxification studies. Metal accumulation in roots and shoots were analyzed and it was observed that roots accumulated a significant amount of Cd (1980 microg g(-1) dry weight), Cr (1540 microg g(-1) dry weight), Cu (1995 microg g(-1) dry weight), and Pb (2040 microg g(-1) dry weight) after 56 d of exposure, though in shoot this was 1110, 618, 795, and 409 microg g(-1) dry weight of Cd, Cr, Cu, and Pb, respectively. In order to assess detoxification mechanisms, non-protein thiols (NP-SH), glutathione (GSH) and phytochelatins (PCs) were analyzed in plants. An increase in the quantity of NP-SH (9.55), GSH (8.30), and PCs (1.25) micromol g(-1) FW were found at 15 microM of Cd, however, a gradual decline in quantity was observed from 15 microM of Cd onwards, after 56 d of exposure. For genotoxicity in plants, cytogenetic end-points such as mitotic index (MI), micronucleus formation (MN), mitotic aberrations (MA) and chromosome aberrations (CA) were examined in root meristem cells of B. juncea. Exposure of Cd revealed a significant (P < 0.05) inhibition of MI, induction of MA, CA, and MN in the root tips for 24 h. However, cells examined at 24 h post-exposure showed concentration-wise recovery in all the endpoints. The data revealed that Indian mustard could be used as a potential accumulator of Cd, Cr, Cu, and Pb due to a good tolerance mechanisms provided by combined/concerted action of NP-SH, GSH, and PCs. Also, exposure of Cd can cause genotoxic effects in B. juncea L. through chromosomal mutations, MA, and MN formation.

Effects of arsenate (AS5+) on growth and production of glutathione (GSH) and phytochelatins (PCS) in Chlorella vulgaris.

The effect of arsenate (As5+) on growth and chlorophyll a production in Chlorella vulgaris, its removal by C. vulgaris and the role of glutathione (GSH) and phytochelatins (PCs) were investigated. C. vulgaris was tolerant to As5+ at up to 200 mg/L and was capable of consistently removing around 70% of the As5+ present in growth media over a wide range of exposure concentrations. Spectral analysis revealed that PCs and their arsenic-combined complexes were absent, indicating that the high bioaccumulation and tolerance to arsenic observed was not due to intracellular chelation. In contrast, GSH was found in all samples ranging from 0.8 mg/L in the control to 6.5 mg/L in media containing 200 mg/L As5+ suggesting that GSH plays a more prominent role in the detoxification of As5+ in C. vulgaris than PC. At concentrations below 100 mg/L cell surface binding and other mechanisms may play the primary role in As5+ detoxification, whereas above this concentration As5+ begins to accumulate inside the algal cells and activates a number of intracellular cell defense mechanisms, such as increased production of GSH. The overall findings complement field studies which suggest C. vulgaris as an increasingly promising low cost As phytoremediation method for developing countries.

The detoxification of lead in Sedum alfredii H. is not related to phytochelatins but the glutathione.

Two ecotypes of S. alfredii [Pb accumulating (AE) and Pb non-accumulating (NAE)] differing in their ability in accumulating Pb were exposed to different Pb levels to evaluate the effects on plant length, photosynthetic pigments, antioxidant enzymes (SOD and APX), cysteine, non-protein thiols (NP-SH), phytochelatins (PCs) and glutathione (GSH) vis-à-vis Pb accumulation. Both ecotypes showed significant Pb accumulation in roots, however only the AE showed significant Pb accumulation in shoots. We found that both AE and NAE of S. alfredii-induced biosynthesis of GSH rather than phytochelatins in their tissue upon addition of even high Pb levels (200 microM). Root and shoot length were mostly affected in both ecotypes after addition of higher Pb concentrations and on longer durations, however photosynthetic pigments did not alter upon addition of any Pb treatment. Both superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities of AE were higher than NAE. The levels of cysteine and NP-SH were also higher in AE than in NAE. Hence, the characteristic Pb accumulation of ecotypes differed presumably in relation to their capacity for detoxification of Pb. These results suggest that enzymatic and non-enzymatic antioxidants play a key role in the detoxification of Pb-induced toxic effects in Sedum alfredii. This plant can be used as an indicator species for Pb contamination.

Thiol metabolism and antioxidant systems complement each other during arsenate detoxification in Ceratophyllum demersum L.

Ceratophyllum demersum L. is known to be a potential accumulator of arsenic (As), but mechanisms of As detoxification have not been investigated so far. In the present study, we analyzed the biochemical responses of Ceratophyllum plants to arsenate (As(V); 0-250 microM) exposure to explore the underlying mechanisms of As detoxification. Plants efficiently tolerated As toxicity up to concentrations of 50 microM As(V) and durations of 4 d with no significant effect on growth by modulating various pathways in a coordinated and complementary manner and accumulated about 76 microg As g(-1)dw. Significant increases were observed in the levels of various thiols including phytochelatins (PCs), the activities of enzymes of thiolic metabolism as well as arsenate reductase (AR). These primary responses probably enabled plants to detoxify at least some part of As(V) through its reduction and subsequent complexation. The maximum proportion of As chelated by PCs was found to be about 30% (at 50 microM As(V) after 2 d). Simultaneously, a significant increase in the activities of antioxidant enzymes was observed and hence plants did not experience oxidative stress when exposed to 50 microM As(V) for 4 d. Exposure of plants to higher concentrations (250 microM As(V)) and/or for longer durations (7 d) resulted in a significant increase in the level of As (maximum 525 microgg(-1)dw at 250 microM after 7 d) and an inverse relationship between As accumulation and various detoxification strategies was observed that lead to enhanced oxidative stress and hampered growth.