GeLC–MS/MS based label free proteomic profiling was used in the large scale identification and quantification of proteins from Brazilian pine (Araucaria angustifolia) embryogenic cell (EC) lines that showed different propensities to form somatic embryos. Using a predicted protein sequence database that was derived from A. angustifolia RNA-Seq data, 2398 non-redundant proteins were identified. The log2 of the spectral count values of 858 proteins of these proteins showed a normal distribution, and were used for statistical analysis. Statistical tests indicated that 106 proteins were significantly differentially abundant between the two EC lines, and that 35 were more abundant in the responsive genotype (EC line SE1) and 71 were more abundant in the blocked genotype (EC line SE6). An increase in the abundance of proteins related to cell defense, anti-oxidative stress responses, and storage reserve deposition was observed in SE1. Moreover, in SE6 we observed an increased abundance of two proteins associated with seed development during the embryogenic cell proliferation stage, which we suggest is associated with genotypes showing a low responsiveness to embryo formation. Differences in protein abundance between the EC lines are discussed in terms of carbohydrate metabolism, cell division, defense response, gene expression, and response to reactive oxygen species.

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.

No abstract

Polyamines (PAs), such as spermidine and spermine, as well as amino acids that are substrates for their biosynthesis, are known to be essential for plant development. However, little is known about the gene expression and metabolic switches associated with the ornithine/arginine and PA biosynthetic pathway during seed development in conifers. To understand these metabolic switches, the enzyme activity of arginine decarboxylase and ornithine decarboxylase, as well as the contents of PAs and amino acids were evaluated in three Araucaria angustifolia (Bertol. Kuntze) seed developmental stages in combination with expression profile analyses of genes associated with the ornithine/arginine and PA biosynthetic pathway. Twelve genes were selected for further analysis and it was shown that the expression profiles of AaADC and AaSAMDC were up-regulated during zygotic embryo development. Polyamines and amino acids were found to accumulate differently in embryos and megagametophytes, and the transition from the globular to the cotyledonary stage was marked by an increase in free and conjugated spermidine and spermine contents. Putrescine is made from arginine, which was present at low content at the late embryogenesis stage, when high content of citrulline was observed. Differences in amino acids, PAs and gene expression profiles of biosynthetic genes at specific seed stages and at each seed transition stage were investigated, providing insights into molecular and physiological aspects of conifer embryogenesis for use in future both basic and applied studies.

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia.