Adaptation of stomatal conductance, photosynthesis and water-use efficiency at shoot and canopy scales in adjacent stands of Dacrycarpus dacrydioides and Podocarpus totara.

We tested an approach to estimate daily canopy net photosynthesis, A, based on estimates of transpiration, E, using measurements of sap flow and water-use efficiency, ω, by measuring δ13C in CO2 respired from shoots in the canopies of two conifers (Podocarpaceae) native to New Zealand. The trees were planted in adjacent 20-year-old stands with the same soil and environmental conditions. Leaf area index was lower for Dacrycarpus dacrydioides D.Don in Lamb (1.34 m2 m-2) than for Podocarpus totara G.Benn. ex D.Don var. totara (2.01 m2 m-2), but mean (± standard error) stem diameters were the same at 152 ± 21 mm for D. dacrydioides and 154 ± 25 mm for P. totara. Over a 28-day period, daily A (per unit ground area) ranged almost five-fold but there were no significant differences between species (mean 2.73 ± 1.02 gC m-2 day-1). This was attributable to higher daily values of E (2.63 ± 0.83 mm day-1) and lower ω (1.35 ± 0.53 gC kg H2O-1) for D. dacrydioides compared with lower E (1.82 ± 0.72 mm day-1) and higher ω (1.90 ± 0.77 gC kg H2O-1) for P. totara. We attributed this to higher nitrogen availability and nitrogen concentration per unit foliage area, Na, and greater exposure to irradiance in the D. dacrydioides canopy compared with P. totara. Our findings support earlier observations that D. dacrydioides is more adapted to sites with poor drainage. In contrast, the high retention of leaf area and maintaining low rates of transpiration by P. totara, resulting in higher water-use efficiency, is an adaptive response to survival in dry conditions. Our findings show that physiological adjustments for two species adapted to different environments led to similar canopy photosynthesis rates when the trees were grown in the same conditions. We demonstrated consistency between whole-tree and more intensive shoot-scale measurements, confirming that integrated approaches are appropriate for comparative estimates of carbon uptake in stands with different species.

Exploring the potential of Canavalia ensiformis for phytoremediation of B10 biodiesel-contaminated soil: insights on aromatic compound degradation and soil fertility.

The widespread use of petroleum-based fuels poses a significant environmental problem due to the risks associated with accidental spills. Among the treatments available, phytoremediation is increasingly accepted as an effective and low-cost solution. This study aimed to evaluate the degradation of the aromatic fraction of biodiesel B10 and the soil fertility analysis in artificially contaminated soils treated with phytoremediation. The experimental design consisted of a 3x3 factorial, with three types of soil treatment: control, autoclaved, and planted with C. ensiformis L, and three levels of B10 biodiesel contamination: 0, 1, and 2%, to simulate spills of 30,000 and 60,000 L ha-1. The soil was analyzed at three depths: 0-10, 10-20, and 20-30 cm. The results indicated that aromatic compound degradation after phytoremediation was superior to 92,76% and 88,65% for 1% and 2% B10 soil contamination, respectively. The fuel contamination affected soil fertility, reducing the availability of phosphorus and zinc while increasing the Total Organic Carbon (TOC), pH, and the availability of manganese and iron for plants.

Numerous studies have been carried out on the phytoremediation of soils contaminated with diesel. However, the potential impact of biodiesel-diesel blends on soil fertility has not been fully explored, leading to a critical knowledge gap. This work investigates the novel effects of biodiesel-diesel blends on phytoremediation and soil fertility, allowing us to expand our understanding of the effectiveness of this approach to remediate contaminated sites.

Electrostatic spraying of imazamox to control the floating aquatic plant Salvinia molesta and its effects on environmental indicators of water quality.

This study aimed to assess, in a microcosm condition, the efficacy of electrostatic spraying of herbicide imazamox in the control of Salvinia molesta and the effects of decomposition of plant material on water quality. The herbicide rates used were 600, 700, 800, and 900 g ai ha-1 and spray volume of 50 L ha-1 in electrostatic application. Control effectiveness was assessed at 7, 15, 30, 45, and 60 days after application (DAA), expressed in percentage (0-100%) of visible injury symptoms in the plants, biochemical oxygen demand (BOD5), chemical oxygen demand (COD), chlorophyll a and pheophytin a contents at 0, 7, 15, 30, 45, and 60 DAA, and fresh and dry biomass at 60 DAA. Imazamox was effective in controlling 63% of S. molesta with 900 g ai ha-1 in 45 DAA and 30% with 800 g ai ha-1 in 30 DAA, and reduced 82.3% and 17.5% of fresh weight and 62.6% and 9.3% of dry weight of plant at 60 DAA, respectively. The imazamox spray reduced chlorophyll a with all doses applied and increased BOD5 in 45 DAA with 900 g ai ha-1 and COD in all assessment periods, but for temperature, dissolved oxygen, electrical conductivity, and pH, there was no significant effect after spraying. The herbicide imazamox reduced S. molesta plants with 900 g ai ha-1, without causing significant effects on environmental indicators of water quality. Electrostatic spraying of herbicide can be used in management strategies of aquatic plants to reduce plant density in water bodies and maintain the colonization of plants at a level not harmful to the aquatic biota.

Assessing the effects of water quality on leaf morphoanatomy, ultrastructure and photosynthetic pigment content of Salvinia auriculata Aubl. (Salviniaceae).

The objective of this study is to evaluate the effects of physical-chemical and biological variables of the water of the Capibaribe River (state of Pernambuco, Brazil) on leaf anatomy, including ultrastructure and photosynthetic pigment of Salvinia auriculata. Specimens of S. auriculata collected in the Gurjaú River, an area with a low pollution degree, were acclimatized in Hoagland's solution and then subjected to three water samples of the Capibaribe River with different levels of pollution. Twenty-one physical-chemical and biological variables were analyzed according to the Standard Methods for the Examination of Water and Wastewater. The results showed that the samples of the Capibaribe River presented nine parameters that did not comply with the current Brazilian legislation. After 15 days of bioassay, S. auriculata presented variations in mesophyll and cuticle thickness, changes in trichome morphology and accumulation of phenolic compounds. No significant differences were observed for photosynthetic pigment content and leaf length of S. auriculata. Multivariate analyses (PCA and Cluster) showed that the point in the Capibaribe River with the highest number of variables that do not comply with the current legislation was responsible for major structural and chemical changes observed in S. auriculata.

Use of Salvinia sp on the adsorption of hexavalent chromium.

The remotion of hexavalent chromium in the form of chromate in aqueous solution was done using the aquatic plant Salvinia sp as biosorbent. The chemical modification of the Salvinia surface was performed by organosolv adapted method. The untreated Salvinia and the modified were characterized by infrared spectroscopy, Boehm titration, scanning electron microscopy, energy dispersive system, point of zero charge, surface area analysis, and porosity. Batch adsorption experiments were performed to observe the effects of pH, contact time, initial concentration, and temperature on the metal removal process. The characterization results show the chemistry modifically changed the modified Salvinia structure compared with untreated Salvinia. The adsorption test results showed the maximum adsorption capacity of 26.03 mg g-1. The kinetic equilibrium was reached in about 3 h, and the better temperature and pH were 298 K and 7, respectively. The adsorption and kinetic models were Freundlich and pseud-second order, respectively. This study showed the Salvinia sp after the chemical treatment can be used with biosorbent for hexavalent chromate in the form of chromate, being a natural material with low cost and plentiful in the environment.

High nitrogen contribution by Gunnera magellanica and nitrogen transfer by mycorrhizas drive an extraordinarily fast primary succession in sub-Antarctic Chile.

Chronosequences at the forefront of retreating glaciers provide information about colonization rates of bare surfaces. In the northern hemisphere, forest development can take centuries, with rates often limited by low nutrient availability. By contrast, in front of the retreating Pia Glacier (Tierra del Fuego, Chile), a Nothofagus forest is in place after only 34 yr of development, while total soil nitrogen (N) increased from near zero to 1.5%, suggesting a strong input of this nutrient. We measured N-fixation rates, carbon fluxes, leaf N and phosphorus contents and leaf δ15 N in the dominant plants, including the herb Gunnera magellanica, which is endosymbiotically associated with a cyanobacterium, in order to investigate the role of N-fixing and mycorrhizal symbionts in N-budgets during successional transition. G. magellanica presented some of the highest nitrogenase activities yet reported (potential maximal contribution of 300 kg N ha-1  yr-1 ). Foliar δ15 N results support the framework of a highly efficient N-uptake and transfer system based on mycorrhizas, with c. 80% of N taken up by the mycorrhizas potentially transferred to the host plant. Our results suggest the symbiosis of G. magellanica with cyanobacteria, and trees and shrubs with mycorrhizas, to be the key processes driving this rapid succession.

Effect of pH on Cr(III) accumulation, biomass production, and phenolic profile in 2 Salvinia species.

We analyzed the effect of pH on Cr(III) accumulation, biomass production, and phenolic profile of Salvinia rotundifolia and Salvinia minima plants grown in the presence of increasing concentrations of CrCl3 . Biomass accumulation, metal tolerance index, and photosynthetic pigment contents indicate that Salvinia rotundifolia seems to be more tolerant of Cr(III) than S. minima at different pHs. Increased metal accumulation by Salvinia species under increasing pH could be explained by changes of the protonation status of cell wall functional groups because both the highest and the lowest pH values used in the present study were outside of the levels at which Cr(III) species start to precipitate. The metal translocation factor indicates that in buffered conditions S. rotundifolia tend to retain more Cr(III) in lacinias than S. minima, probably through the involvement of insoluble phenolics. The results of the present study could be useful to the management of solution pH to maximize the removal of Cr(III) by aquatic plants. Environ Toxicol Chem 2019;38:167-176. © 2018 SETAC.

Accumulation of copper by the aquatic macrophyte Salvinia biloba Raddi (Salviniaceae) / Acumulação de cobre pela macrófita aquática Salvinia biloba Raddi (Salviniaceae)

Abstract Aquatic macrophytes have properties and mechanisms which are useful for the removal of substances in solution, commonly used in phytoremediation processes in aquatic environments. This study evaluated the performance of copper (Cu) accumulation by Salvinia biloba Raddi (Salviniaceae) in different metal concentrations (1, 3 and 5 µg mL-1), as well as the control treatment, measured at intervals of 0, 7 and 14 days under laboratory conditions, with control as to pH and luminosity. After the experiment, the S. biloba biomass was washed, kiln dried, crushed and subjected to the process of digestion, and subsequently the accumulated copper content was determined by atomic absorption spectroscopy. The results showed that S. biloba is apt at accumulating copper, varying significantly between different treatments and days of exposure to the contaminant, as well as its interaction (treatment × days). The highest accumulation values were observed in treatment with 5 µg mL-1, which at 14 days, with 11,861 µg g-1 of copper. We observed symptoms of toxicity and mortality in plants, probably indicating the effect of copper on the species when at high levels. Salvinia biloba is an efficient species in the removal of copper in solution, its recommendation as a remediating agent in aquatic ecosystems being possible.

Resumo Macrófitas aquáticas apresentam propriedades e mecanismos úteis à remoção de substâncias presentes em solução, comumente, utilizadas em processos de fitorremediação de ambientes aquáticos. Este estudo avaliou o desempenho de acumulação de cobre (Cu) por Salvinia biloba Raddi (Salviniaceae) em diferentes concentrações do metal (1, 3 e 5 µg mL-1), além do tratamento controle, avaliados em intervalos de 0, 7 e 14 dias, sob condições laboratoriais, com controle de pH e luminosidade. Após o experimento a biomassa de S. biloba, foi lavada, seca em estufa, triturada e submetida ao processo de digestão, sendo, posteriormente, o teor de cobre acumulado determinado por espectroscopia de absorção atômica. Os resultados evidenciaram que S. biloba é hábil em acumular cobre em sua biomassa, variando significativamente entre os diferentes tratamentos e dias de exposição ao contaminante, bem como por sua interação (tratamentos × dias). Os maiores teores de acúmulo foram observados no tratamento com 5 µg mL-1, que aos 14 dias apresentou até 11.861 µg g-1 de Cu. Observamos sintomas de toxicidade e mortalidade nas plantas, evidenciando, provavelmente, o efeito do cobre sobre a espécie quando em elevados níveis. Salvinia biloba é uma espécie eficiente na remoção do cobre em solução, sendo possível sua recomendação como agente de remediação em ecossistemas aquáticos.

Accumulation of copper by the aquatic macrophyte Salvinia biloba Raddi (Salviniaceae).

Aquatic macrophytes have properties and mechanisms which are useful for the removal of substances in solution, commonly used in phytoremediation processes in aquatic environments. This study evaluated the performance of copper (Cu) accumulation by Salvinia biloba Raddi (Salviniaceae) in different metal concentrations (1, 3 and 5 µg mL-1), as well as the control treatment, measured at intervals of 0, 7 and 14 days under laboratory conditions, with control as to pH and luminosity. After the experiment, the S. biloba biomass was washed, kiln dried, crushed and subjected to the process of digestion, and subsequently the accumulated copper content was determined by atomic absorption spectroscopy. The results showed that S. biloba is apt at accumulating copper, varying significantly between different treatments and days of exposure to the contaminant, as well as its interaction (treatment × days). The highest accumulation values were observed in treatment with 5 µg mL-1, which at 14 days, with 11,861 µg g-1 of copper. We observed symptoms of toxicity and mortality in plants, probably indicating the effect of copper on the species when at high levels. Salvinia biloba is an efficient species in the removal of copper in solution, its recommendation as a remediating agent in aquatic ecosystems being possible.

Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia.

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.

Elucidation of the polyamine biosynthesis pathway during Brazilian pine (Araucaria angustifolia) seed development.

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.

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized.

Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae).

Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 µM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants.

Lead accumulation reduces photosynthesis in the lead hyper-accumulator Salvinia minima Baker by affecting the cell membrane and inducing stomatal closure.

Salvinia minima Baker accumulates a fair amount of lead in its tissues; however, no studies have investigated the effect of lead on the physiological processes that affect photosynthesis in this species. The objective of the present study was to assess whether the high amounts of lead accumulated by S. minima can affect its photosynthetic apparatus. The physiological changes in the roots and leaves in response to lead accumulation were analyzed. An exposure to 40 µM Pb(NO3)2 for 24 h (first stage) was sufficient to reduce the photosynthetic rate (Pn) by 44%. This reduction in Pn was apparently the result of processes at various levels, including damage to the cell membranes (mainly in roots). Interestingly, although the plants were transferred to fresh medium without lead for an additional 24 h (second stage), Pn not only remained low, but was reduced even further, which was apparently related to stomatal closure, and may have led to reduced CO2 availability. Therefore, it can be concluded that lead exposure first decreases the photosynthetic rate by damaging the root membrane and then induces stomatal closure, resulting in decreased CO2 availability.

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.

Control of resin production in Araucaria angustifolia, an ancient South American conifer.

Araucaria angustifolia is an ancient slow-growing conifer that characterises parts of the Southern Atlantic Forest biome, currently listed as a critically endangered species. The species also produces bark resin, although the factors controlling its resinosis are largely unknown. To better understand this defence-related process, we examined the resin exudation response of A. angustifolia upon treatment with well-known chemical stimulators used in fast-growing conifers producing both bark and wood resin, such as Pinus elliottii. The initial hypothesis was that A. angustifolia would display significant differences in the regulation of resinosis. The effect of Ethrel(®) (ET - ethylene precursor), salicylic acid (SA), jasmonic acid (JA), sulphuric acid (SuA) and sodium nitroprusside (SNP - nitric oxide donor) on resin yield and composition in young plants of A. angustifolia was examined. In at least one of the concentrations tested, and frequently in more than one, an aqueous glycerol solution applied on fresh wound sites of the stem with one or more of the adjuvants examined promoted an increase in resin yield, as well as monoterpene concentration (α-pinene, ß-pinene, camphene and limonene). Higher yields and longer exudation periods were observed with JA and ET, another feature shared with Pinus resinosis. The results suggest that resinosis control is similar in Araucaria and Pinus. In addition, A. angustifolia resin may be a relevant source of valuable terpene chemicals, whose production may be increased by using stimulating pastes containing the identified adjuvants.

Sulfur metabolism: different tolerances of two aquatic macrophytes exposed to arsenic.

The toxicity of arsenic (As) and the mechanisms of response to this pollutant were analyzed in two aquatic plant species, one sensitive and one tolerant to the pollutant, Salvinia minima and Lemna gibba, respectively. The plants, grown in nutrient solution at pH 6.5, were exposed to As concentrations of 0.0 and 1.0mgL(-1) for 3 days. Both species accumulated As in their tissues, which resulted in increases in H2O2 production. L. gibba accumulated eleven times more As than S. minima. However, L. gibba was more tolerant, as shown by the absence of cell membrane damage and, despite greater accumulation, smaller growth reduction than S. minima. Indeed, the index of tolerance to As was twenty percent higher in L. gibba than in S. minima, which most likely results from the presence of a more efficient defense system. This defense system in L. gibba is most likely based on sulfate absorption, assimilation and metabolism. L. gibba showed an increase in sulfate absorption and adenosine-5'-triphosphate (ATP) sulfurylase activity (the first enzyme of the inorganic sulfate assimilation pathway) following exposure to As. Consequently, the plant produced greater concentrations of sulfur-containing compounds that are involved in cellular detoxification, such as glutathione and non-protein thiols, and demonstrated greater enzymatic activity of γ-glutamylcysteine synthetase, glutathione S-transferase and glutathione reductase. Therefore, the plant׳s ability to increase absorption, assimilation and metabolism of sulfur are key steps for tolerance to oxidative stress triggered by metals.

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.

Immunoanalysis of dehydrins in Araucaria angustifolia embryos.

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos, a species of endangered and economically important conifers, native to southern Brazil, northeastern Argentina, and eastern Paraguay. The A. angustifolia seeds have been categorized as recalcitrant. Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment, a highly conserved lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10 kDa, was present in the cotyledons only. Following alkaline phosphatase (AP) treatment, a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation. Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy. At the subcellular level, dehydrins were immunolocalized in the nuclei, protein bodies, and microbodies. In the nucleus, dehydrins were found to be associated with chromatin. We concluded that the gel mobility shift for the four main bands (probably due to phosphorylation), the presence of thermosensitive bands, and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this species.

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.