Proteomic analysis and polyamines, ethylene and reactive oxygen species levels of Araucaria angustifolia (Brazilian pine) embryogenic cultures with different embryogenic potential

Somatic embryogenesis is an important biotechnological tool in the large-scale propagation of elite genotypes and ex situ conservation of conifer species. Protocols for the induction and proliferation of embryogenic cultures (ECs) of Brazilian pine (Araucaria angustifolia (Bert.) O. Ktze) are well established, although the proper formation of mature somatic embryos (SEs) is still problematic. Thus, the identification of molecular markers for the screening of ECs able to respond to maturation conditions (abscisic acid and osmotic agents) is highly desirable. To develop molecular markers for the early detection of ECs able to develop well-formed SEs under maturation conditions, we analyzed the proteins found during the proliferation phase of A. angustifolia cell lines with different embryogenic capabilities, with one cell line being responsive to maturation conditions (R cell line), and one cell line that presented blocked development of SEs (B cell line). In addition, based on the peptides identified, polyamine levels (free and conjugate), ethylene production and reactive oxygen species (ROS) emission were analyzed using both EC lines (R and B cell lines). A marked difference in the biochemistry of ECs between these two cell lines was observed. Eleven proteins that were differentially expressed in the cell lines were identified by the combination of two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry. Among these, S-adenosylmethionine synthase, the enzyme associated with polyamines and ethylene biosynthesis, was observed exclusively in the R cell line, while a protein linked to the oxidative stress subunit F of NADH dehydrogenase was observed exclusively in the B cell lines. Additionally, B cell lines showed higher levels of diamine putrescine and lower levels of ethylene. Higher values of ethylene and ROS were observed for the cell line that showed normal development of SEs. Altogether, our results open new perspectives in the optimization of culture conditions for A. angustifolia somatic embryogenesis, as well as establishing biochemical markers for the early selection of ECs during maturation trials.This research was carried out with financial support from the State of Sao Paulo Research Foundation (FAPESP) (10/50868- 9), the National Council for Scientific and Technological Development (CNPq), the Coordination of Personal Higher Education Improvement (CAPES) (02437/09-0) and PETROBRAS (SAP 4600279702)