Cold stress on Araucaria angustifolia embryogenic cells results in oxidative stress and induces adaptation: implications for conservation and propagation.

Araucaria angustifolia (Bert.) O. Kuntze is a species critically endangered of extinction and its development and propagation is strongly affected by abiotic stress. We have previously shown the activation of uncoupling protein in A. angustifolia embryogenic stem cells subjected to cold stress. Now, we have furthered those studies by exposing these cells to cold stress (4 ± 1 °C for either 24 or 48 h) and evaluating parameters associated with oxidative stress and alterations in the cellular and mitochondrial responses. Cold stress affect the H2O2 levels and lipid peroxidation increased after both stress condition, an effect associated with the decrease in the activities of peroxidases, catalase and ascorbate/dehydroascorbate ratio. On the other hand, the activities of ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases increased as an indication of adaptation. Another important impact of cold stress conditions was the decrease of external alternative NAD(P)H dehydrogenases activity and the increase of mitochondrial mass. These results show that cold stress induces oxidative stress in A. angustifolia embryogenic cells, which results in activation of the glutathione-ascorbate cycle as a compensation for the decrease in the activities of catalase, peroxidases, and external NAD(P)H dehydrogenases. Our results contribute to the understanding of the pathways that gymnosperms employ to overcome oxidative stress, which must be explored in order to improve the methods of conservation and propagation of A. angustifolia.

Polyamines affect the cellular growth and structure of pro-embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines.

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro-embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal-bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H(+) -ATPase P- and V-types and H(+) -PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.

Structural and component characterization of meristem cells in Araucaria angustifolia (Bert.) O. Kuntze zygotic embryo.

Araucaria angustifolia, the Brazilian pine, is an endangered native conifer with economic and ecological importance. The female cone develops seeds containing the zygotic embryo, which, at cotyledonary stage, shows well-developed meristems. Little is known about the structure of gymnosperm meristems. In the present work, the composition and morphological organization of Araucaria angustifolia shoot and root apical meristems were studied during embryo development, using histochemical and microscope analyses. Histochemical evaluation revealed the presence of cellulose within the cell wall, cells with the presence of total proteins that react with Coomassie Brilliant Blue, starch grains, and large nuclei with evident nucleoli in the cytoplasm. Scanning electron microscopy showed apical meristem surface morphology, and both scanning and transmission microscopy revealed a thin and irregular cell wall with plasmodesmata and within the cells, mitochondria, many vacuoles, lipid bodies, Golgi bodies, and many amyloplasts with endoplasmic reticulum surrounding them and large nuclei. Similar to angiosperm cells, A. angustifolia meristem cells exhibit pluripotent characteristics, such as apparatus for intercellular communication and differentiation.

Arabinogalactan-proteins from cell suspension cultures of Araucaria angustifolia.

Arabinogalactan-proteins (AGPs), found in the culture medium of suspension cells of Araucaria angustifolia grown in plant growth regulator-free and plant growth regulator-containing BM media, BM0 and BM2, respectively, were evaluated quantitatively and qualitatively. The concentrated extracellular fractions (CEFs), obtained from suspension cell cultures grown for 20 days in BM0 and BM2 media yielded two fractions, CEF-0 and CEF-2, respectively. CEF-0 and CEF-2 was submitted to selective precipitation using the beta-glucosyl Yariv reagent (beta-GlcY) to isolate AGPs for structural characterization; this yielded fractions designated CEF-0YPF and CEF-2YPF, respectively. The monosaccharide composition analysis established that samples were composed of Rha, Ara, Gal and uronic acid in a molar ratio 3:37:55:5 (CEF-0YPF) and 1:37:58:4 (CEF-2YPF), although trace amounts (<0.5 mol%) of Xyl were also found. Methylation analysis of CEF-YPF fractions showed similar results for both CEF-0YPF and CEF-2YPF, with non-reducing terminal units of Araf, Arap, Galp, Rhap and Xylp, as well as 3-O-substituted and 5-O-substituted Araf units and 3-O-substituted, 6-O-substituted and 3,6-di-O-substituted Galp units. The amino acid composition analysis established Ser, Ala, and Hyp as major amino acids in both samples. In conclusion, this investigation has shown that CEF-0YPF and CEF-2YPF contain macromolecules having typical AGP characteristics, including a Hyp/Ala/Ser-rich protein moiety, a (1-->3) and/or (1-->6) linked beta-d-galactopyranosyl main chain substituted by Gal, Ara, Rha and Xyl residues, and binding affinity for beta-GlcY and monoclonal anti-AGP antibodies.

Development of mucilage cells of Araucaria angustifolia (Araucariaceae).

The roles of mucilage cells were investigated through morphological and cytological analysis during leaf development in young Araucaria angustifolia plants. Differentiation began in leaf primordia in the shoot apex, when the young cells underwent a greater increase in volume in comparison with other mesophyll cells. The mucilage polysaccharides were synthesized by dictyosomes, from where they were taken by large vesicles and released into a cavity formed by detachment of the tonoplast, which was separated from the cytoplasm. At the end of differentiation, the cell was completely filled with mucilage, a gel consisting of a denser reticular structure surrounding less dense regions. The nucleus and cytoplasm were degenerated in mature cells. The A. angustifolia mucilage cells presented some cytological resemblances to the mucilage cells of members of some dicotyledonous families; however, differences in the dictyosomes and the secretion route were observed. Translocation and water storage of solutes was suggested by the use of the hydroxy pyrenetrisulfonic acid tri-sodium salt apoplastic tracer. The tonoplast detachment, dechromatinization, nuclear condensation, and general degeneration of the membrane systems observed during maturity indicated a programmed cell death process, one not yet described for angiosperm mucilage cells.