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.

Functions of stone cells and oleoresin terpenes in the conifer defense syndrome.

Conifers depend on complex defense systems against herbivores. Stone cells (SC) and oleoresin are physical and chemical defenses of Sitka spruce that have been separately studied in previous work. Weevil oviposit at the tip of the previous year's apical shoot (PYAS). We investigated interactions between weevil larvae and trees in controlled oviposition experiments with resistant (R) and susceptible (S) Sitka spruce. R trees have an abundance of SC in the PYAS cortex. SC are mostly absent in S trees. R trees and S trees also differ in the composition of oleoresin terpenes. Transcriptomes of R and S trees revealed differences in long-term weevil-induced responses. Performance of larvae was significantly reduced on R trees compared with S trees under experimental conditions that mimicked natural oviposition behavior at apical shoot tips and may be attributed to the effects of SC. In oviposition experiments designed for larvae to feed below the area of highest SC abundance, larvae showed an unusual feeding behavior and oleoresin appeared to function as the major defense. The results support a role for both SC and oleoresin terpenes and possible synergies between these traits in the defense syndrome of weevil-resistant Sitka spruce.

Foliar epidermal micromorphology and its taxonomic implications in some selected species of Athyriaceae.

For the robust identification of taxonomically complex fern family like Athyriaceae, light and scanning electron microscopy is significance implications. This article present first microscopic investigation of foliar micromorphology of 3 genera and 10 species belonging to Athyriaceae namely, Athyrium, Deparia, and Diplazium were collected from different localities in Malakand Division, Northern Pakistan. In present study we compare foliar micromorphology of all 10 species using standard protocols of light microcopy (LM) and scanning electron microscopy. Qualitative micromorphological variations in shape of epidermal cells, anticlinal wall pattern, stomatal type and shape, stomatal pore shape, guard cells shape, and trichomes types were studied. In addition, some quantitative characters were also studied and data were statistically analyzed in epidermal cell size, stomatal size, stomatal pore size, stomatal density, and stomatal index. The pivotal result of study include; shape of epidermal cell in all species is irregular on both abaxial and adaxial surfaces. The anticlinal walls are sinuous in most of the species but some species have irregular lobed and broadly lobed wall. Leaves are hypostomatic in all studied species. Two main categories of stomatal type were found: polocytic and anomocytic. Unicellular nonglandular trichomes were observed in only one species Athyrium mackinnoni. The variation in foliar micromorphological characters between the genera and within the species was useful in identification and classification and have potential taxonomic significance for species differentiation. An identification key using micromorphological characters are provided to distinguish genera and species.

Cell size and wall dimensions drive distinct variability of earlywood and latewood density in Northern Hemisphere conifers.

Interannual variability of wood density - an important plant functional trait and environmental proxy - in conifers is poorly understood. We therefore explored the anatomical basis of density. We hypothesized that earlywood density is determined by tracheid size and latewood density by wall dimensions, reflecting their different functional tasks. To determine general patterns of variability, density parameters from 27 species and 349 sites across the Northern Hemisphere were correlated to tree-ring width parameters and local climate. We performed the same analyses with density and width derived from anatomical data comprising two species and eight sites. The contributions of tracheid size and wall dimensions to density were disentangled with sensitivity analyses. Notably, correlations between density and width shifted from negative to positive moving from earlywood to latewood. Temperature responses of density varied intraseasonally in strength and sign. The sensitivity analyses revealed tracheid size as the main determinant of earlywood density, while wall dimensions become more influential for latewood density. Our novel approach of integrating detailed anatomical data with large-scale tree-ring data allowed us to contribute to an improved understanding of interannual variations of conifer growth and to illustrate how conifers balance investments in the competing xylem functions of hydraulics and mechanical support.

Xylogenesis: Coniferous Trees of Temperate Forests Are Listening to the Climate Tale during the Growing Season But Only Remember the Last Words!

The complex inner mechanisms that create typical conifer tree-ring structure (i.e. the transition from large, thin-walled earlywood cells to narrow, thick-walled latewood cells) were recently unraveled. However, what physiological or environmental factors drive xylogenesis key processes remain unclear. Here, we aim to quantify the influence of seasonal variations in climatic factors on the spectacular changes in the kinetics of wood cell differentiation and in the resulting tree-ring structure. Wood formation was monitored in three sites over 3 years for three coniferous species (Norway spruce [Picea abies], Scots pine [Pinus sylvestris], and silver fir [Abies alba]). Cell differentiation rates and durations were calculated and related to tracheid final dimensions and corresponding climatic conditions. On the one hand, we found that the kinetics of cell enlargement and the final size of the tracheids were not explained by the seasonal changes in climatic factors. On the other hand, decreasing temperatures strongly constrained cell wall deposition rates during latewood formation. However, the influence of temperature was permanently written into tree-ring structure only for the very last latewood cells, when the collapse of the rate of wall deposition was no longer counterbalanced by the increase of its duration. Our results show that the formation of the typical conifer tree-ring structure, in normal climatic conditions, is only marginally driven by climate, suggesting strong developmental control of xylogenesis. The late breakage of the compensatory mechanism at work in the wall deposition process appears as a clue to understand the capacity of the maximum latewood density to record past temperature conditions.

Formation of vacuolar tannin deposits in the chlorophyllous organs of Tracheophyta: from shuttles to accretions.

Most Tracheophyta synthesize-condensed tannins (also called proanthocyanidins), polymers of catechins, which appear in the vacuole as uniformly stained deposits-termed tannin accretions-lining the inner face of the tonoplast. A large body of evidence argues that tannins are formed in recently described thylakoid-derived organelles, the tannosomes, which are packed in membrane-bound shuttles (Brillouet et al. 2013); it has been suggested that shuttles agglomerate into tannin accretions. The aim of the study was to describe the ontogenesis of tannin accretions in members of the Tracheophyta. For this purpose, fresh specimens of young tissues from diverse Tracheophyta were cut, gently lacerated in paraformaldehyde, and examined using light, epifluorescence, confocal, and transmission electron microscopy. Fresh samples were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Our observations showed that vacuolar accretions (1 → 40 µm), that constitute the typical form of tannin storage in tannin-producing Tracheophyta, are formed by agglomeration (not fusion) of shuttles containing various proportions of chlorophylls and tannins.

Toxic effects of mercury on PSI and PSII activities, membrane potential and transthylakoid proton gradient in Microsorium pteropus.

Mercury (Hg) is one of the top toxic metals in environment and it poses a great risk to organisms. This study aimed to elucidate the toxic effects of Hg(2+) on energy conversion of photosystem I (PSI) and photosystem II (PSII), membrane potential and proton gradient of Microsorium pteropus (an aquatic plant species). Contents of chlorophyll a, chlorophyll b and carotenoids, quantum yield and electron transfer of PSI and PSII of M. pteropus exposed to various concentrations of Hg(2+) were measured. With increasing Hg(2+) concentration, quantum yield and electron transport of PSI [Y(I) and ETR(I)] and PSII [Y(II) and ETR(II)] decreased whereas limitation of donor side of PSI [Y(ND)] increased. At ⩾165µgL(-1) Hg(2+), quantum yield of non-light-induced non-photochemical fluorescence quenching in PSII [Y(NO)] significantly increased but quantum yield of light-induced non-photochemical fluorescence quenching [Y(NPQ)] decreased. Membrane potential (Δψ) and proton gradient (ΔpH) of M. pteropus were reduced significantly at 330µg L(-1) Hg(2+) compared to control. Mercury exposure affected multiple sites in PSII and PSI of M. pteropus.

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.

Cryopreservation of Tetraclinis articulata (vahl.) Masters.

Tetraclinis articulata shoot tips excised from in vitro grown shoots were cryopreserved using a modified PVS2-based vitrification protocol. Preliminary experiments with non-cryostored shoot tips showed that the high concentrations of sucrose in loading (LS), vitrification (PVS2) and unloading (US) solutions employed in the protocol were very toxic for the explants. Replacement of sucrose by sorbitol in equal molar concentration in all these solutions enhanced survival of shoot tips after all treatments. However, cold-hardening of donor shoots before shoot tip excision was strictly required to obtain post-rewarming survival. Therefore, the protocol was outlined as follows: pre-conditioning of explants at 4 degree C for 3 weeks in the dark; excision of 1 mm long shoot tips; loading for 20 min in modified LS at room temperature; dehydration in modified PVS2 at 0 degree C for 60 min; immersion in liquid nitrogen (LN); rewarming at 40 degree C for 2 min and subsequent transfer of shoot tips in modified US for 20 min.

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.

Terpenoid biosynthesis and specialized vascular cells of conifer defense.

Defense-related terpenoid biosynthesis in conifers is a dynamic process closely associated with specialized anatomical structures that allows conifers to cope with attack from many potential pests and pathogens. The constitutive and inducible terpenoid defense of conifers involves several hundred different monoterpenes, sesquiterpenes and diterpenes. Changing arrays of these many compounds are formed from the general isoprenoid pathway by activities of large gene families for two classes of enzymes, the terpene synthases and the cytochrome P450-dependent monooxygenases of the CYP720B group. Extensive studies have been conducted on the genomics, proteomics and molecular biochemical characterization of these enzymes. Many of the conifer terpene synthases are multi-product enzymes, and the P450 enzymes of the CYP720B group are promiscuous in catalyzing multiple oxidations, along homologous series of diterpenoids, from a broad spectrum of substrates. The terpene synthases and CYP720B genes respond to authentic or simulated insect attack with increased transcript levels, protein abundance and enzyme activity. The constitutive and induced oleoresin terpenoids for conifer defense accumulate in preformed cortical resin ducts and in xylem trauma-associated resin ducts. Formation of these resin ducts de novo in the cambium zone and developing xylem, following insect attack or treatment of trees with methyl jasmonate, is a unique feature of the induced defense of long-lived conifer trees.

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.

[Molecular-biological nature of morphological abnormalities induced by chronic irradiation in coniferous plants from the Chernobyl nuclear power plant exclusion zone: emphasis on a possible role of the cytoskeleton]. / Molekuliarno-biologicheskaia priroda morfologicheskikh anomalii sredi golosemennykh rastenii, indutsirovannykh khronicheskim oblucheniem v zone otchuzhdeniia Chernobyl'skoi AES: aktsent na vozmozhnuiu rol' tsitoskeleta.

Results of the analysis of morphological abnormalities in coniferous plants from the Chernobyl exclusion zone are described. It is shown that such features as total and individual protein content, genome organization and peculiarities of its expression, and karyotype are different for the control and morphologically abnormal needles. A possible role of the cytoskeleton structures in the abnormal morphogenesis in plants is discussed.

Disruption of cellulose synthesis by isoxaben causes tip swelling and disorganizes cortical microtubules in elongating conifer pollen tubes.

In elongating pollen tubes of the conifer Picea abies (Norway spruce), microtubules form a radial array beneath the plasma membrane only at the elongating tip and an array parallel with elongation throughout the tube. Tips specifically swell following microtubule disruption. Here we test whether these radial microtubules coordinate cell wall deposition and maintain tip integrity as tubes elongate. Control pollen tubes contain cellulose throughout the walls, including the tip. Pollen tubes grown in the presence of isoxaben, which disrupts cellulose synthesis, are significantly shorter with a decrease in cellulose throughout the walls. Isoxaben also significantly increases the frequency of tip swelling, with no effect on tube width outside of the swollen tip. The decrease in cellulose is more pronounced in pollen tubes with swollen tips. The effects of isoxaben are reversible. Following isoxaben treatment, the radial array of microtubules persists beneath the plasma membrane of nonswollen tips, while this array is specifically disrupted in swollen tips. Microtubules instead form a random network throughout the tip. Growth in these pollen tubes is turgor driven, but the morphological changes due to isoxaben are not just the result of weakened cell walls since pollen tubes grown in hypoosmotic media are not significantly shorter but do have swollen tips and tubes are wider along their entire length. We conclude that the radial microtubules in the tip do maintain tip integrity and that the specific inhibition of cellulose microfibril deposition leads to the disorganization of these microtubules. This supports the emerging model that there is bidirectional communication across the plasma membrane between cortical microtubules and cellulose microfibrils.