ABSTRACT
Cysteine S-nitrosation is a redox-based post-translational modification that mediates nitric oxide (NO) regulation of various aspects of plant growth, development and stress responses. Despite its importance, studies exploring protein signaling pathways that are regulated by S-nitrosation during somatic embryogenesis have not been performed. In the present study, endogenous cysteine S-nitrosation site and S-nitrosated proteins were identified by iodo-TMT labeling during somatic embryogenesis in Brazilian pine, an endangered native conifer of South America. In addition, endogenous -S-nitrosothiol (SNO) levels and S-nitrosoglutathione reductase (GSNOR) activity were determined in cell lines with contrasting embryogenic potential. Overall, we identified an array of proteins associated with a large variety of biological processes and molecular functions with some of them already described as important for somatic embryogenesis (Class IV chitinase, pyruvate dehydrogenase E1 and dehydroascorbate reductase). In total, our S-nitrosoproteome analyses identified 18 endogenously S-nitrosated proteins and 50 in vitro S-nitrosated proteins (after GSNO treatment) during cell culture proliferation and embryo development. Furthermore, SNO levels and GSNOR activity were increased during embryo formation. These findings expand our understanding of the Brazilian pine proteome and shed novel insights into the potential use of pharmacological manipulation of NO levels by using NO inhibitors and donors during somatic embryogenesis.
ABSTRACT
The mechanisms that control polyamine (PA) metabolism in plant cell lines with different embryogenic potential are not well understood. This study involved the use of two Araucaria angustifolia cell lines, one of which was defined as being blocked, in that the cells were incapable of developing somatic embryos, and the other as being responsive, as the cells could generate somatic embryos. Cellular PA metabolism was modulated by using 5 mM arginine (Arg) or ornithine (Orn) at two time points during cell growth. Two days after subculturing with Arg, an increase in citrulline (Cit) content was observed, followed by a higher expression of genes related to PA catabolism in the responsive cell line; whereas, in the blocked cell line, we only observed an accumulation of PAs. After 14 d, metabolism was directed towards putrescine accumulation in both cell lines. Exogenous Arg and Orn not only caused a change in cellular contents of PAs, but also altered the abundance of a broader spectrum of amino acids. Specifically, Cit was the predominant amino acid. We also noted changes in the expression of genes related to PA biosynthesis and catabolism. These results indicate that Arg and Orn act as regulators of both biosynthetic and catabolic PA metabolites; however, we suggest that they have distinct roles associated with embryogenic potential of the cells.
