The metabolic response of Araucaria angustifolia embryogenic cells to heat stress is associated with their maturation potential.

Araucaria angustifolia is a critically endangered species and its distribution can be affected by an increase in temperature. In this study, we evaluated the effects of heat stress (30°C) on Araucaria angustifolia cell lines responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The viability of both cell lines was reduced by heat stress and mitochondria were the organelles most affected. Heat stress for 24h increased the reactive oxygen species (ROS) levels in SE1 cells, followed by a reduction at 48 and 72h. In SE6 cells, an increase occurred after 24 and 48h of stress, returning to control levels at 72h. H2 O2 levels were increased after 24h for both SE1 and SE6 cells, being higher for SE6. Interestingly, at 48 and 72h, H2 O2 levels decreased in SE1 cells, while in SE6, the values returned to the control levels. The respiration of SE6 cells in the presence of oxidisable substrates was inhibited by heat stress, in agreement with the high lipid peroxidation levels. The AaSERK1 gene was identified in both cultures, with greater expression in the SE1 line. Heat stress for 24 and 48h increased gene expression only in this cell line. The activity of peroxidase, superoxide dismutase and enzymes of the glutathione/ascorbate cycle was increased in both cell lines subjected to heat stress. Catalase activity was increased only in SE6 cells at 72h of exposure. These results show that responsive SE1 cells can modulate ROS levels more efficiently than SE6 when these cells are stressed by heat. This ability may be related to the maturation capacity of these cells.

Mitochondrial bioenergetics and enzymatic antioxidant defense differ in Paraná pine cell lines with contrasting embryogenic potential.

Araucaria angustifolia is classified as a critically endangered species by the International Union for Conservation of Nature. This threat is worsened by the inefficiency of methods for ex-situ conservation and propagation. In conifers, somatic embryogenesis (SE) associated with cryopreservation is an efficient method to achieve germplasm conservation and mass clonal propagation. However, the efficiency of SE is highly dependent on genotype responsivity to the artificial stimulus used in vitro during cell line proliferation and later during somatic embryo development. In this study, we evaluated the activity of antioxidant enzymes and characterized mitochondrial functions during the proliferation of embryogenic cells of A. angustifolia responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The activities of the antioxidant enzymes GR (EC 1.6.4.2), MDHAR (EC 1.6.5.4), and POX (EC 1.11.1.7) were increased in SE1 culture, while in SE6 culture, only the activity of DHAR (EC 1.8.5.1) was significantly higher. Additionally, SE6 culture presented a higher number of mitochondria, which agreed with the increased rate of oxygen consumption compared to responsive SE1 culture; however, the mitochondrial volume was lower. Although the ATP levels did not differ, the NAD(P)H levels were higher in SE1 cells. NDs, AOX, and UCP were less active in responsive SE1 than in non-responsive cells. Our results show significant differences between SE1 and SE6 embryogenic cells regarding mitochondrial functions and antioxidant enzyme activities, which may be intrinsic to the in vitro proliferation phase of both cell lines, possessing a crucial role for the induction of in vitro maturation process.

Plant Proteomics and Systems Biology.

Proteome analysis of model and non-model plants is a genuine scientific field in expansion. Several technological advances have contributed to the implementation of different proteomics approaches for qualitative and quantitative analysis of the dynamics of cellular responses at the protein level. The design of time-resolved experiments and the emergent use of multiplexed proteome analysis using chemical or isotopic and isobaric labeling strategies as well as label-free approaches are generating a vast amount of proteomics data that is going to be essential for analysis of protein posttranslational modifications and implementation of systems biology approaches. Through the target proteomics analysis, especially the ones that combine the untargeted methods, we should expect an improvement in the completeness of the identification of proteome and reveal nuances of regulatory cellular mechanisms related to plant development and responses to environmental stresses. Both genomic sequencing and proteomic advancements in the last decades coupled to integrative data analysis are enriching biological information that was once confined to model plants. Therewith, predictions of a changing environment places proteomics as an especially useful tool for crops performance.

Polyamine- and Amino Acid-Related Metabolism: The Roles of Arginine and Ornithine are Associated with the Embryogenic Potential.

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.

Quantitative proteomic analysis of Araucaria angustifolia (Bertol.) Kuntze cell lines with contrasting embryogenic potential.

GeLC­MS/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.

Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission.

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia.

Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine).

Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.

Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia.

Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.