Microsatellite development for the relictual conifer Araucaria araucana (Araucariaceae) using next-generation sequencing

? Premise of the study: In this study, the 454 GS-FLX genome sequence system was used for the identification and characterization of microsatellites in Araucaria araucana, one of the most important and endangered species of Chilean and Argentinean native forests. ? Methods and Results: A total of 35876 reads were identified, 96% of which were within the size range selected for further analysis. Of these, 1563 contained a microsatellite insert suitable for primer design. Ten simple sequence repeat (SSR) markers provided easily interpretable patterns and were used to evaluate the genetic diversity in four populations of the species. The 10 microsatellites showed high polymorphism levels, with a total of 99 alleles and 32 private alleles. The observed heterozygosity was high and ranged from 0.513 to 0.723. ? Conclusions: The primers presented in this study display high genetic diversity and may provide useful information for the design of conservation strategies in Araucaria araucana.     

由国分类学文献中南洋杉属两种之订正

通过调查,对Araucaria cunninghamii和A.heterophylla的选模式和中国分类学文献(如<广州植物志>、<中国树木学>、<中国高等植物图鉴>、<中国植物志>、<中国树木志>、、<中国高等植物>)的A.cunninghamii和A.heterophylla(或A.excelsa)作比较,证实中国分类学文献中的A.cunninghamii和A.heterophylla(或A.excelsa)分别是A.heterophylla(Salisb.)Franco和A.cunninghamii Aiton ex A.Cunn..这两种植物的叶形变化较大,但有明显的区别:诺福克南洋杉A.heterophylla幼树的末级小枝的叶细长呈线形、叶尖急尖,牛长角小于45°,腹面无明显的脊,成龄株的末级小枝的叶扁平呈鳞片状;南洋杉A.cunninghamii幼树的末级小枝的叶呈两侧扁的钻形、叶尖短刺状,生长角45°-90°,腹背两面具明显的脊,成龄株的末级小枝的叶呈四棱状钻形.     

Tree-ring responses in Araucaria araucana to two major eruptions of Lonquimay Volcano (Chile)

Palaeoclimate proxies have demonstrated links between climate changes and volcanic activity. However, not much is known about the impact of volcanic eruptions on forest productivity. Here we used tree-ring width and annually resolved carbon and oxygen isotopic records from tree rings of Araucaria araucana (Molina) K. Koch, providing a centennial-scale reconstruction of tree ecophysiological processes in forest stands nearby the Lonquimay Volcano (Chile). We observed a mean decrease in tree-ring width following the major eruption of 1988–1990 (with aerosol emission), most probably caused by the modified ecological conditions due to acid rain and ash deposition, while a generally negative relationship between δ¹³C and δ¹⁸O would point to a decline in humidity and precipitation. More negative δ¹³C and lower δ¹⁸O values (positive correlation) following the major eruption of 1887–1890 (without aerosol emission) would suggest high stomatal conductance and moisture availability, though tree-ring width (and probably photosynthetic rate) was unaltered. At least for this sample of trees, in the case of eruption with large tephra emission, the beneficial effect of aerosol light scattering on tree productivity appears to be outweighed by the detrimental effect of eruption-induced toxic deposition. Signals of the two major eruptions of the past 200?years at Lonquimay were present in tree rings of nearby A. araucana. No unique response of tree functions to volcanic eruptions can be expected, but rather (1) the variable volcanic properties and (2) the complex interplay of diffuse light increase (aerosol scattering), air temperature decrease (cloud shading), and toxic deposition impact (volcanic ash), makes any prediction of tree growth and ecophysiological response very challenging.     

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.     

Purification of a debranching enzyme (R-enzyme) from malted barley,and the role of the enzyme in the digestion of starch granules during the germination of barley seeds

A debranching enzyme (R-enzyme or pullulan-6-glucanohydrolase, EC3.2.1.41), free from contaminating carbohydrases and homogeneous by poly(acrylamide) disc-gel electrophoresis, has been purified from malted barley. A partially purified preparation of this enzyme (3.1 units/mg of protein) accelerated the rate of digestion of barley-starch granules by the action of purified alpha and beta amylases to the same extent as was effected by the dialyzed, crude extract from malted barley. Contrary to expectation, the debranching enzyme, purified to homogeneity (10 units/mg of protein), had very little accelerating effect. These results indicate that a factor or factors, which may be maltase or α-d-glucosidase and were lost during the purification of the debranching enzyme, may play a role in the digestion of starch granules by the dialyzed, crude extract from malted barley in vitro and by enzymes in the endosperm of germinating barley seeds in vivo. The debranching enzymes, including barley-malt R-enzyme, Aerobacter pullulanase, and Pseudomonas isoamylase, did not digest starch granules to a detecble extent.     

Purification and biological effects of Araucaria angustifolia (Araucariaceae) seed lectin

This paper describes the purification and characterization of a new N-acetyl-d-glucosamine-specific lectin from Araucaria angustifolia (AaL) seeds (Araucariaceae) and its anti-inflammatory and antibacterial activities. AaL was purified using a combination of affinity chromatography on a chitin column and ion exchange chromatography on Sephacel-DEAE. The pure protein has 8.0kDa (SDS-PAGE) and specifically agglutinates rabbit erythrocytes, effect that was independent of the presence of divalent cations and was inhibited after incubation with glucose and N-acetyl-d-glucosamine. AaL showed antibacterial activity against Gram-negative and Gram-positive strains, shown by scanning electron microscopy. AaL, intravenously injected into rats, showed anti-inflammatory effect, via carbohydrate site interaction, in the models of paw edema and peritonitis. This lectin can be used as a tool for studying bacterial infections and inflammatory processes.     

Isolation and characterization of microsatellite markers for Araucaria angustifolia (Araucariaceae)

Araucaria angustifolia is a dioecious tree species that occurs in the southern part of Brazil. Because of the intense exploitation of the species due to its valuable wood, only 2% of the original population still remains. Twenty‐nine species‐specific and highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. Levels of polymorphism were evaluated using a total of 16 adult trees from a natural population. An average of 8.1 alleles per locus was detected, and expected heterozygosity ranged from 0.63 to 0.72.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

AFLP markers provide insights into the evolutionary relationships and diversification of New Caledonian Araucaria species (Araucariaceae)

? Premise of the study: Despite its small size, New Caledonia is characterized by a very diverse flora and striking environmental gradients, which make it an ideal setting to study species diversification. Thirteen of the 19 Araucaria species are endemic to the territory and form a monophyletic group, but patterns and processes that lead to such a high species richness are largely unexplored. ? Methods: We used 142 polymorphic AFLP markers and performed analyses based on Bayesian clustering algorithms, genetic distances, and cladistics on 71 samples representing all New Caledonian Araucaria species. We examined correlations between the inferred evolutionary relationships and shared morphological, ecological, or geographic parameters among species, to investigate evolutionary processes that may have driven speciation. ? Key results: We showed that genetic divergence among the present New Caledonian Araucaria species is low, suggesting recent diversification rather than pre-existence on Gondwana. We identified three genetic groups that included small-leaved, large-leaved, and coastal species, but detected no association with soil preference, ecological habitat, or rainfall. The observed patterns suggested that speciation events resulted from both differential adaptation and vicariance. Last, we hypothesize that speciation is ongoing and/or there are cryptic species in some genetically (sometimes also morphologically) divergent populations. ? Conclusions: Further data are required to provide better resolution and understanding of the diversification of New Caledonian Araucaria species. Nevertheless, our study allowed insights into their evolutionary relationships and provides a framework for future investigations on the evolution of this emblematic group of plants in one of the world's biodiversity hotspots.     

The steam volatile oil of Wollemia nobilis and its comparison with other members of the Araucariaceae (Agathis and Araucaria)

The leaf essential oil of Wollemia nobilis (Wollemi Pine) has been investigated and compared with other members of the family Araucariaceae. All araucaroids examined yielded steam volatile oils in low yields. The oil from Wollemia nobilis was composed mainly of (+)-16-kaurene (60%), together with alpha-pinene (9%) and germacrene-D (8%). Oils from Agathis species endemic to Australia were high in monoterpenes, in contrast to those isolated from extra-Australian species. The major constituents of A. atropurpurea oil were phyllocladene (13%) and 16-kaurene (19%), followed by alpha-pinene (8%) and delta-cadinene (9%). A. microstachya yielded oil in which alpha-pinene (18%) was the major component; the only other components in excess of 5% were myrcene (7%), bicyclogermacrene (6%) and delta-cadinene (6%). A. robusta oil contained spathulenol (37%) and rimuene (6%). Approximately 40% of the oil was unidentified sesquiterpenes. A. australis oil contained 16-kaurene (37%), sclarene (5%) and an unidentified oxygenated diterpene K (12%) as major components; the only other compound in excess of 5% was germacrene-D (9%). 5,15-Rosadiene (60%), and 16-kaurene (7%) were the major constituents of A. macrophylla oil. A. moorei oil was rich in sesquiterpenes, but the only compounds in excess of 5% were allo-aromadendrene (6%), germacrene-D, delta-cadinene (10%), an unidentified sesquiterpene (12%) and 16-kaurene (6%). In A. ovata oil the most significant compounds were caryophyllene oxide (15%) and phyllocladene (39%). Araucaria angustifolia contained germacrene-D (9%) and the diterpenes hibaene (30%) and phyllocladene (20%) as major components of its essential oil. Oils of A. bidwillii, A. columnaris and A. cunninghamii were all low in mono- and sesquiterpenes and high in diterpenes. In the first, hibaene (76%) was the major constituent; the second contained hibaene (9%), sclarene (6%), luxuriadiene (13-epi-dolabradiene)(23%) and two unidentified diterpene hydrocarbons (B) (33%) and (E) (10%). In the last, 16-kaurene (53%) was the most significant component followed by hibaene (29%). A. heterophylla was unusual in that over half the oil was made up of the monoterpenoid alpha-pinene (52%), with phyllocladene (32%) being the only other compound of significance. alpha-Pinene (18%) was a significant component of A. hunsteinii oil; sclarene (11%) and germacrene-D (5%) were the only other compounds present in concentrations of more than 5%. A. luxurians oil was composed of 5,15-rosadiene (20%) and luxuriadiene (13-epi-dolabradiene) (66%), previously unreported from natural sources. The major components of A. montana were phyllocladene (61%) and 16-kaurene (23%). Sclarene (20%), luxuriadiene (19%) and the unidentified diterpene hydrocarbons (B) (25%) and (E) (10%) were the most important constituents of A. muelleri oil. A. scopulorum contained large amounts of 16-alpha-phyllocladanol (41%) as well as luxuridiene (10%) and delta-cadinene and alpha-copaene, both at 6%.     

Structure of mixed ombrophyllous forests with Araucaria angustifolia (Araucariaceae) under external stress in Southern Brazil

This study is part of the Floristic and Forest Inventory of Santa Catarina, conceived to evaluate forest resources, species composition and structure of forest remnants, providing information to update forest conservation and land use policy in Southern Brazilian State of Santa Catarina (95 000 km2). In accordance to the Brazilian National Forest Inventory (IFN-BR), the inventory applies systematic sampling, with 440 clusters containing four crosswise 1 000m2 plots (20 x 50m) each, located on a 10 x 10km grid overlaid to land use map based on classification of SPOT-4 images from 2005. Within the sample units, all woody individuals of the main stratum (DBH > or = 10cm) are measured and collected (fertile and sterile), if not undoubtedly identified in field. Regeneration stratum (height > 1.50m; DBH < 10cm) is registered in 100m2 in each sample unit. Floristic sampling includes collection of all fertile trees, shrubs and herbs within the sample unit and in its surroundings. This study performs analysis based on 92 clusters measured in 2008 within an area of 32320km2 of mixed ombrophyllous forests with Araucaria angustifolia located at the state's high plateau (500m to 1 560m above sea level at 26 degrees 00'-28 degrees 30' S and 49 degrees 13'-51 degrees 23' W). Mean density (DBH > or = 10cm) is 578 individuals/ha (ranging from 85/ha to 1 310/ha), mean species richness in measured remnants is 35 (8 to 62), Shannon and Wiener diversity index (H') varies between 1.05 and 3.48. Despite high total species diversity (364 Magnoliophyta, five Coniferophyta and one tree fern) and relatively high mean basal area (25.75m2/ha, varying from 3.87 to 68.85m2/ha), the overwhelming majority of forest fragments are considered highly impacted and impoverished, mostly by logging, burning and extensive cattle farming, turning necessary more efficient protection measures. Basal area was considered an appropriate indicator for stand quality and conservation status.     

The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase

High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.     

Reproductive biology of the “Brazilian pine” (Araucaria angustifolia – Araucariaceae): Development of microspores and microgametophytes

Araucaria angustifolia Bert. (O. Ktze), also known as the “Brazilian pine”, is native to the South of Brazil. This species has a long reproductive cycle, taking about 29–34 months. Its cones begin to develop in early January and remain dormant from March to July. In August, they become active again and microsporogenesis occurs, which proceeds until September. From September to October, microgametogenesis is established and pollination occurs from October to November. Meiosis is asynchronous, with simultaneous cytokinesis, and the tetrads are of the tetrahedral and isobilateral type. During gametogenesis, microgametophytes gradually develop an axial row of cells that are isolated by internal callose and undergo four mitotic cycles until pollen dispersal. In mature pollen grains, the vegetative cells do not possess a cell wall, but maintain strong internal polarization. The pollen of A. angustifolia is suboblate, without apertures or air sacs. Histochemical analysis of the sporoderm was also performed and when compared to other conifer families, showed the most simplified intine structure among the group. Embryological characteristics analyzed during the phenological phases of this species showed certain peculiarities, knowledge about which may be helpful contributing to the management and conservation of A. angustifolia.     

New insights into the genetic structure of Araucaria araucana forests based on molecular and historic evidences

The conservation of genetic resources is a prerequisite for the maintenance of long-lived forest species. Araucaria araucana (Mol.) K. Koch is one of the oldest conifers in South America and a representative symbol of Chilean forest due to its endemicity and longevity. The aim of this study was to evaluate the genetic structure of the current A. araucana populations in Chile, to verify the possible genetic divergence between Coastal and Andean populations and to assess whether bottleneck events have influenced habitat fragmentation and threaten the genetic resources and evolutionary potential of the species. Twelve natural populations, nine from the Andes Cordillera and three from the Coast Cordillera were analysed by means of eight genomic microsatellite markers developed in A. araucana. Results of analysis of molecular variance (AMOVA) highlighted significant differentiation between Coastal and Andean populations (16 %; P?=?0.004), detecting one significant barrier that separated populations from both Cordilleras as maximally differentiated areas. At local scale, both ranges revealed significant inter-population differentiation, with higher values for Coastal populations compared with Andean populations. These results suggested the presence of four gene pools (three in the Andes and one in the Coast Cordilleras) and one population (VIL) in the Coast Cordillera that differed to the rest. The differentiation between the Andean and Coastal populations may provide important baseline data that should allow further studies of landscape genetics in the species and that can contribute to develop conservation strategies for its genetic resources.     

Genetic structure of Araucaria angustifolia (Araucariaceae) populations in Brazil: implications for the in situ conservation of genetic resources

The distribution of the genetic variation within and among natural populations of A. ANGUSTIFOLIA growing in different regions in Brazil was assessed at microsatellite and AFLP markers. Both markers revealed high gene diversity ( H = 0.65; AR = 9.1 for microsatellites and H = 0.27; P = 77.8 % for AFLPs), moderate overall differentiation ( RST = 0.13 for microsatellites and FST = 0.10 for AFLPs), but high divergence of the northernmost, geographically isolated population. In a Bayesian analysis, microsatellite data suggested population structure at two levels: at K = 2 and at K = 3 in agreement to the geographical distribution of populations. This result was confirmed by the UPGMA dendrogram based on microsatellite data (bootstrap support > 95 %). Non-hierarchical AMOVA revealed high variation among populations from different A POSTERIORI defined geographical groups. The genetic distance between sample locations increased with geographical distance for microsatellites ( R = 0.62; P = 0.003) and AFLPs ( R = 0.32; P = 0.09). This pattern of population differentiation may be correlated with population history such as geographical isolation and postglacial colonization of highlands. Implications of the population genetic structure for the conservation of genetic resources are discussed.     

Effect of drought on proteins and isoenzymes in rice during germination

Two drought tolerant varieties TKM-1 and TKM-2 and two drought susceptible varieties Jaya and Improved Sabarmati of rice were studied for soluble protein pattern and isoenzymes of malate dehydrogenase, glutamate dehydrogenase, esterase and peroxidase during germination at different water stress. MDH, GDH and esterase patterns were not affected, but the soluble proteins were changed. Peroxidase isoenzyme pattern from drought tolerant and susceptible varieties showed characteristic differences. The intensity of bands with higher electrophoretic mobility decreased in Jaya and Improved Sabarmati while in TKM-1 and TKM-2 the intensity of these bands did not change much after 72 hr water stress. In shoots of Jaya and Improved Sabarmati, the activity of the peroxidase isoenzymes decreased more than in TKM-1 and TKM-2 shoots with increase in water stress.