The role of Phytochelatin Synthase in the microalga Scenedesmus acutus M. (Sphaeropleales).

The role of Phytochelatin Synthase in the microalga Scenedesmus acutus M. (Sphaeropleales) M. Ferrari, R. Cozza, M. Marieschi, R. Ruotolo, A. Torelli Phytochelatins (PCs) are small cysteine-rich peptides that are not genetically encoded but are synthesized by Phytochelatin Synthase (PCS) in the presence of glutathione as substrate. The constitutive expression of PCS and the presence of homologues of the PCS gene(s) in plants growing in ecosystems geographically remote from metal-contaminated sites, as well as in representatives of various kingdoms of living organisms, suggest that PCS has a wide range of different functions (Clemens et al. 2009). However, the main function of PCs in plants is to immobilize, sequester, and detoxify metal ions (Shukla et al. 2013). The effect of metals on the activity of PCS can differ not only between species, but also between different isoforms of the enzyme within a species. Although it is evident that chromium (Cr) ions can stimulate the formation of PCs in plants (Yu et al. 2018), there is no information on their presence in algae in response to Cr exposure. In this study, two strains of the green alga Scenedesmus acutus with different Cr(VI) sensitivity, namely the wild type (wt) and the chromium-tolerant strain (Cr-t), were selected as a model algal species to increase the knowledge on the role of PCs in Cr responses in microalgae. We previously reported the first evidence for a PCS gene (SaPCS) from the microalga S. acutus. More recently, we have also identified two SaPCS isoforms due to a putative alternative splicing. To define the role of PCS in Cr detoxification, we analyzed the levels of SaPCS transcription, and the abundance of PCS by using RT-qPCR and Western blot, respectively, in both strains of S. acutus after 24h culture in standard and in Cr supplemented medium (1 and 2 mg Cr (VI)/l). Given the relationship between sulfur (S) metabolism and Cr(VI) tolerance in S. acutus (Sardella et al. 2019), we also performed the same analyses in S-replete cells of both strains after medium renewal following S-starvation. The results showed a different behavior between strains in the stress response. RT-qPCR analysis revealed an increase in SaPCS transcription after Cr(VI) stress and after medium renewal following S-starvation in wt cells. Instead, the Cr-t strain induced SaPCS transcription only under S-replete conditions. Western blot analysis, performed with a polyclonal antibody raised against PCS of Arabidopsis thaliana, revealed an immunoreaction signal on two proteins with molecular weights of approximately 73 and 37 kDa. While the abundance of the 73 kDa protein did not change after the stresses studied in the two strains, the 37 kDa protein increased after S-starvation in both strains but in a significant manner only in wt. Finally, a preliminary quantification of PC production was performed, through LC Mass analysis in the two strains in preculture with or without S and under 1 mg Cr(VI)/l, which showed no modulation of PC abundance in response to Cr(VI) stress and/or S deprivation. Nevertheless, the Cr-t strain maintained PCs level significantly higher than the wt at the end of S-starvation. These observations suggest that PCS in S. acutus may not be involved in Cr(VI) detoxification, but may play a role in intracellular sulfur balance or in the cell homeostasis during stress. The PC production in the other experimental conditions (i.e. exposures longer than those studied and different Cr(VI) concentrations) should be investigated to better clarify the role of the enzyme in Cr(VI) detoxification. Letteratura citata Shukla D., Tiwari M., Tripathi E.D., Nath P., Trivedi P.K. (2013). Synthetic phytochelatins complement a phytochelatin-deficient Arabidopsis mutant and enhance the accumulation of heavy metal(loid)s. Biochem Biophy Res Commun 434:664–669 Clemens S., Peršoh D. (2009). Multi-tasking phytochelatin synthases. Plant Sci., 177, 266–271. Yu, X.Z., Ling, Q.L., Li, Y.H., Lin Y.J. (2018) mRNA Analysis of Genes Encoded with Phytochelatin Synthase (PCS) in Rice Seedlings Exposed to Chromium: The Role of Phytochelatins in Cr Detoxification. Bull Environ Contam Toxicol 101, 257–261. Sardella A., Marieschi M., Mercatali I., Zanni C., Gorbi G., Torelli A. (2019). The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase. Aquatic Toxicology, 216:105320. AUTORI Michele Ferrari (michele.ferrari@unical.it), Radiana Cozza, Department of Biology, Ecology and Earth Science, University of Calabria, Via P. Bucci, 87036 Rende (CS). Matteo Marieschi, Roberta Ruotolo, Anna Torelli, Department of Life Sciences, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma