Rapid High Quality RNA Preparation from Pine Seedlings

Conventional RNA extraction methods have been shown to produce poor quality RNA when applied to pine and other gymnosperms. We present a protocol for extracting highly pure RNA from pine. Modifications to conventional procedures include: 1) the use of seedlings, 2) the use of phenol and PVP to rapidly remove DNA, proteins and pigments, and 3) the use of salt precipitation to remove other contaminating compounds. The procedure can be completed in less than eight hours. Yield and purity were monitored by denaturing gel electrophoresis and by UV absorbance (A₂₆₀ /A₂₈₀ and A₂₆₀/A₂₃₀). These ratios were over 2, indicating an absence of contaminating metabolites. Additionally, a new absorbance ratio (A₂₆₀/A₂₁₀) was introduced to monitor the RNA purity in each step (it indicates the ratio of covalent links in the solution belonging to RNA). The yield was around 300 µg total RNA per gram of tissue of Pinus sylvestris and over 400 µg of total RNA per gram of P. pinaster tissue, which is a high recovery (more than 63%) for gymnosperms. The RNA was of sufficient quality for use in a RT-PCR reaction that amplified 1 kb of the pine GS gene. This protocol has been applied with success to other woody plants like Populus species.Abbreviations: DEPC, diethyl pyrocarbonate; AMV, avian myeloblastosis virus; FW, fresh weight.     

Cavitation fatigue in conifers: a study on eight European species

After drought-induced embolism and repair, tree xylem may be weakened against future drought events (cavitation fatigue). As there are few data on cavitation fatigue in conifers available, we quantified vulnerability curves (VCs) after embolism/repair cycles on eight European conifer species. We induced 50% and 100% loss of conductivity (LC) with a cavitron, and analyzed VCs. Embolism repair was obtained by vacuum infiltration. All species demonstrated complete embolism repair and a lack of any cavitation fatigue after 50% LC . After 100% LC, European larch (Larix decidua), stone pine (Pinus cembra), Norway spruce (Picea abies), and silver fir (Abies alba) remained unaffected, while mountain pine (Pinus mugo), yew (Taxus baccata), and common juniper (Juniperus communis) exhibited 0.4–0.9 MPa higher vulnerability to embolism. A small cavitation fatigue observed in Scots pine (Pinus sylvestris) was probably biased by incomplete embolism repair, as indicated by a correlation of vulnerability shifts and conductivity restoration. Our data demonstrate that cavitation fatigue in conifers is species-specific and depends on the intensity of preceding LC. The lack of fatigue effects after moderate LC, and relevant effects in only three species after high LC, indicate that conifers are relatively resistant against cavitation fatigue. This is remarkable considering the complex and delicate conifer pit architecture and may be important considering climate change projections.     

Induction of conifer immune responses by phytopathogenic fungus metabolites

The elicitor activity of compounds extracted from the mycelia of six species of phytopathogenic fungi was assessed from the sizes of necrotic lesions on the external surface of the living trunk phloem of five coniferous species inhabiting Siberia: Siberian larch (Larix sibirica L.), Scotch pine (Pinus sylvestris L.), Siberian spruce (Picea obovata Ledeb.), Siberian fir (Abies sibirica L.), and cedar pine (Pinus sibirica (Rupr.) Mayr.). The compounds for inoculation were extracted from the mycelium of ascomycetes imperfect, and basidium fungi; the living mycelia of these fungi were also used. The fungal extract or mycelium was placed into the hollows 7 mm in diameter in the trunk bark. Infection triggered the formation of hypersensitivity necrotic lesions in the inner bark exceeding in size those appeared after control wounding of four tree species (larch, pine, spruce, and cedar); fir was an exclusion. In experiments with tree trunks and conifer calluses, a dependence of immune response parameters (the sizes of necrotic lesions and the content of lignin and bound proanthocyanidins) on the quantity of the fungal preparation was elucidated. The largest necrotic lesions appeared after injection of 500μg of the fungal preparation into the hollow in the trunk, and its higher quantities did not increase the indices measured. The size of the necrotic lesion on the trunk bark is supposed to be used as a promising index characterizing the level of tree immunity and tolerance under various ecological conditions.     

Differential selection of North American and Scandinavian conifer browse by northwestern moose (Alces alces andersoni) in winter

Scandinavian moose (Alces alces) eat Scots pine (Pinus sylvestris) in winter. Although North American moose are known to eat conifers such as true firs (Abies spp.) in winter, substantial consumption of pine by moose in North America has not been documented. Here, we document short-term winter preferences of human-habituated northwestern moose (Alces alces andersoni) for branches of mature North American and European conifer species as determined by a cafeteria-style feeding trial. Moose selected for species such as Douglas fir (Pseudotsuga menziesii; from which they took the smallest bite diameters) while avoiding species such as lodgepole pine (Pinus contorta; from which they took the largest bites) and hybrid white spruce (Picea glauca × engelmanii). The amount of species-specific biomass consumed by moose was negatively correlated with bite diameters taken from branches of those species and did not appear to be significantly influenced by differences in twig morphology between species. Our trial suggests that northwestern moose readily consume conifers in winter and, from the species we tested, prefer Douglas fir. While no clear preference existed between Scots pine and lodgepole pine, moose avoided lodgepole pine, but not Scots pine, relative to Douglas fir. Our trial suggests that northwestern moose are more likely to feed on the branches of Douglas fir than pine, which may be of interest to foresters managing conifers within the North American range of moose, particularly where Scots pine are being considered for planting.     

Accumulation of Phenolic Compounds in the Necrotic Areas of Austrian and Red Pine Needles Due to Salt Spray

Austrian pine (Pinus nigra) and red pine (P resinosa) were testedfor needle damage caused by salt spray. Morphology and histologyof newly grown needles were compared in 3- and 6-year-old plantsgrowing in the greenhouse under controlled conditions. Pottedplants covered with plastic bags were sprayed daily either withdistilled H₂O or saturated salt solution. Needle tips, mid-partsand bases were fixed after 1, 3 and 24 h, and 3, 7 and 30 din glutaraldehyde+0·1 % caffeine for precipitation ofphenolics. Histochemical reactions and electron microscope observationsshowed that the morphological damage is linked to increasedphenolic compound deposition in mesophyll cells, beginning directlyunder the stomata. Disorganization of membranes leads to deathof the mesophyll cells, even before morphological damage isrecognizable as brown spots on the needle surface. Thus thehistochemical reactions and observations of cell structure revealeddamage earlier than macroscopic observations. Salt injury, phenolic compounds, histological damage, ultrastructural damage, salt spray, Pinus nigra, Austrian pine, Pinus resinosa, red pine     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

长白山北坡不同林型内红松年表特征及其与气候因子的关系

运用树木年轮学的基本原理和方法,选取了长白山北坡保存完好的典型性植被阔叶红松林,探讨了杨桦红松林和椴树红松林内建群种红松(Pinus koraiensis)径向生长对气候要素的响应.结果表明,长白山北坡红松的径向生长对降水较为敏感,杨桦红松林和椴树红松林中,红松年轮宽度均与当年7月以及上一年9月的降水呈显著正相关关系.不同林型内红松的生长与气候因子的关系也有差异.椴树红松的年轮宽度还与上年7月的降水显著负相关,与当年3、4月份的平均气温呈显著正相关.而杨桦红松林内红松年轮宽度和平均温度没有显著的相关关系.特征年分析进一步验证了响应函数相关分析的结果,即当年生长季以及上年生长季末的降水充足促进了红松的径向生长;椴树红松林中,初春温度的升高有利于红松的生长.     

Establishment of

We compared establishment of Douglas fir (Pseudotsuga menziesii) and Corsican pine (Pinus nigra) seedlings in kanuka (Kunzea ericoides) and manuka (Leptospermum scoparium) shrubland to test the hypothesis that Douglas fir, because of its greater shade tolerance, is better able to establish in woody communities than pine species. Seed of the conifer species was sown under a range of canopy covers at six sites, the cover being low-statured vegetation in openings between stands, stand edges, and moderate and dense canopies. After three growing seasons, survival of Corsican pine seedlings was greatest in the open and declined progressively as canopy cover increased. This contrasted with Douglas fir, where survival was greatest at the canopy edge. Survival of Douglas fir seedlings significantly exceeded that of Corscican pine seedlings under dense canopy positions. Seedling numbers of both species declined significantly with increasing leaf area index of manuka, but not kanuka stands, where seedling numbers were lower. Leaf area index of manuka stands accounted for substantially greater variation in number and survival of Corsican pine than Douglas fir seedlings. It is concluded that Douglas fir is better able to establish in shaded environments in woody communities than Corsican pine; however, further monitoring is required to confirm the long-term survival of both species under the moderate and dense canopy positions in this trial.     

Parameter-specific hydroclimatic sensitivity of a low-elevation network of living and historical tree-ring series from north-eastern Austria

Compared to the alpine regions of Austria, the eastern part of the country is overall warmer and drier with some tree species growing at the dry limit within their ecological range. This suggests that tree rings may be a valuable proxy for hydroclimatic variability. In this study, we develop ring-width, earlywood-width and latewood-width chronologies obtained from drought sensitive living trees and historical timber from one of the driest and warmest regions of Austria, the Weinviertel. For this, samples of four different tree species − fir (Abies alba Mill.), spruce (Picea abies (L.) Karst.), oak (Quercus petraea Liebl., Q. robur L., Q. cerris L.), and pine (Pinus sylvestris L., P. nigra Arnold) were collected from 88 sites (23 forest stands and 65 historical constructions). 1589 samples (oak: 592 samples, pine: 577, spruce: 212, fir: 208) were dated. Dendro-provenancing was required to ensure that only regional material has entered the chronologies. According to the analyses, historical pine and oak wood were nearly 100% regional, while spruce and fir wood were imported to a great extent with only 44% spruce and 35% fir confirmed to be regional. Because site conditions of historical wood samples are unknown but can have a significant influence on tree growth, changes to site replication over time were important in the assessment of chronology reliability. We also controlled for potential artificial increases in sample depth when more than one construction element is made from a single trunk.We assessed the pine (1584-2011 AD) and oak (1244-2011 AD) chronologies’ potential for reconstructing past hydroclimatic variability by means of response functions in a 17-months window from previous June to current October with climate data from three weather stations (Vienna, Retz, Brno) from 1897 to 2010 AD. The highest seasonal response coefficients are for oak and pine ring width (∼0.43) with respect to aggregated March to July precipitation totals and Thornthwaite climatic water balance. These chronologies reveal a high potential for estimating past changes in regional-scale moisture availability during the earlier growing season.     

千烟洲针叶林的比叶面积及叶面积指数

根据实测数据计算了湿地松(Pinus elliotii)、马尾松(P. massoniana) 和杉木(Cunninghamia lanceolata)不同年龄、不同类型叶片的生物量和比叶面积,并结合样地调查数据和相对生长方程计算了中国科学院千烟洲试验站20年生湿地松林、马尾松林、杉木林和针叶混交林的叶面积指数。根据拟合结果,选择如下方程计算3个树种的叶生物量:湿地松W=12.074 1D^{2.151 5}、马尾松W=6.972 7D^{2.197 3}和杉木W=5.261 9D^{2.302 7}。湿地松林的叶生物量(0.822 kg·m^-2)最大,其次为针叶混交林(0.679 kg·m^-2),马尾松林和杉木林相差不大(分别为林0.528和0.572 kg·m^-2)。不同树种、不同年龄、不同类型叶片的比叶面积比较发现,新叶的比叶面积大于老叶,三针一束叶的比叶面积略大于两针一束叶,马尾松的平均半比表面积(8.62 m²·kg^-1)大于湿地松(6.04 m²·kg^-1)和杉木(7.91 m²·kg^-1)。胸径与单木叶片半表面积之间的经验方程为:湿地松LA=0.073D^{2.151 5}、马尾松LA=0.060D^{2.197 3}和杉木LA=0.042D^{2.302 7}。据此计算湿地松林的叶面积指数为5.03,马尾松林和杉木林为4.31,针叶混交林为4.77,该结果比利用CI-110植被冠层数字图像仪测得的结果偏大。     

Transgenic Christmas Trees Regenerated from Agrobacterium tumefaciens-mediated Transformation of Zygotic Embryos Using the Green Fluorescence Protein as a Reporter

Mature zygotic embryos of recalcitrant Christmas tree species Fraser fir [Abies fraseri (Pursh) Poir], and Nordmann fir (Abies nordmanniana L.k.), and Virginia pine (Pinus virginiana Mill.) were used as explants for Agrobacterium tumefaciens strain GV3850-mediated transformation using the gfp (green fluorescent protein) gene as a reporter. Factors including media used for inoculation and co-cultivation, concentrations of acetosyringone, and antibiotics in tissue culture media have been evaluated. A high transformation frequency was obtained on TE medium containing 50μM acetosyringone and using 500 mg/l timentin to eliminate bacteria. Transient gene expression was observed in all three Christmas tree species, but transgenic plants were only produced from Virginia pine. Stable integration and expression of transgenes in the plant genome of Virginia pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable transformation system has been established in Virginia pine and this system would provide an opportunity to transfer economically important genes into Christmas tree species.     

Factors affecting the extraction of intact ribonucleic Acid from plant tissues containing interfering phenolic compounds

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions.     

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified as Methylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.     

Large‐scale assessment of regeneration and diversity in Mediterranean planted pine forests along ecological gradients

Aim There is increasing concern regarding sustainable management and restoration of planted forests, particularly in the Mediterranean Basin where pine species have been widely used. The aim of this study was to analyse the environmental and structural characteristics of Mediterranean planted pine forests in relation to natural pine forests. Specifically, we assessed recruitment and woody species richness along climatic, structural and perturbation gradients to aid in developing restoration guidelines. Location Continental Spain. Methods We conducted a multivariate comparison of ecological characteristics in planted and natural stands of main Iberian native pine species (Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus nigra and Pinus sylvestris). We fitted species‐specific statistical models of recruitment and woody species richness and analysed the response of natural and planted stands along ecological gradients. Results Planted pine forests occurred on average on poorer soils and experienced higher anthropic disturbance rates (fire frequency and anthropic mortality) than natural pine forests. Planted pine forests had lower regeneration and diversity levels than natural pine forests, and these differences were more pronounced in mountain pine stands. The largest differences in recruitment – chiefly oak seedling abundance – and species richness between planted and natural stands occurred at low‐medium values of annual precipitation, stand tree density, distance to Quercus forests and fire frequency, whereas differences usually disappeared in the upper part of the gradients. Main conclusions Structural characteristics and patterns of recruitment and species richness differ in pine planted forests compared to natural pine ecosystems in the Mediterranean, especially for mountain pines. However, management options exist that would reduce differences between these forest types, where restoration towards more natural conditions is feasible. To increase recruitment and diversity, vertical and horizontal heterogeneity could be promoted by thinning in high‐density and homogeneous stands, while enrichment planting would be desirable in mesic and medium‐density planted forests.     

The multivariate underpinnings of recruitment for three Pinus species in montane forests of the Sierra Nevada,USA

Recruitment success can have significant consequences on forest community dynamics. Seedlings and saplings were studied for 3 five-needled white pine species: sugar pine (Pinus lambertiana), western white pine (P. monticola), and whitebark pine (P. albicaulis) across an elevational gradient, representing three distinct forest types, in the Sierra Nevada. This study was designed to determine the importance of local environmental and biological conditions versus broad regional climate patterns influencing conifer recruitment. No relationships were found between recruitment and precipitation, but increasing nighttime temperature was positively correlated with sugar pine and western white pine recruitment and negatively correlated with whitebark pine recruitment. Recruitment pulses were found for sugar pine and western white pine from 1995 to 2006. Whitebark pine recruitment is low and relatively steady from 1970 to 2000 with no observed new recruits after 2001. Source strength was the most consistent predictor of white pine recruitment along with soil properties, climate, light, and the presence of non-conspecifics. Source, light, and soil explained 71 % of the variation in sugar pine recruitment. The presence of non-conspecifics, source, and climate explained 71 % of the variation in western white pine recruitment, and the presence of non-conspecifics and source explained 36 % of the variation for whitebark pine. Recruitment present in forest communities of the Sierra Nevada have successfully transitioned through critical stages (i.e., seed and germinant) and appear to be influenced by multiple factors at both local and regional scales.     

Decline and Death of Pinus spp. in Delaware Caused by Bursaphelenchus xylophilus

Etiological studies to determine the cause of decline and death of Pinus spp. in Delaware were initiated in 1980. The pinewood nematode, Bursaphelenchus xylophilus, was found to be the major canse of mortality in Japanese black pine (Pinus thunbergii). When inoculated into healthy 5-yr-old Japanese black pines, B. xylophilus produced typical decline symptoms observed in the field. The xylophilous fungi most often associated with declining trees, Rhizosphaera pini, Fusarium spp., and Pestalotia funerea, were not pathogenic to Japanese black pine in greenhouse tests. Mineral analyses of soil and foliage showed no significant differences between healthy and infested trees. B. xylolyhilus was also found on loblolly pine (P. taeda), scrub pine (P. virginiana), Scots pine (P. sylvestris), red pine (P. resinosa), Eastern white pine (P. strobus), and pitch pine (P. rigida).     

万仙山油松径向生长与气候因子的关系

以万仙山油松(Pinus tabulaeformis)为样本,建立了油松树木年轮宽度序列的标准年表(STD)。与附近气象因子的相关结果表明:与当年6月平均温度呈较为显著的负相关,与前一年的9月和当年的5月以及春季(3-5月)的降水量呈显著正相关,显然影响万仙山油松生长的主要气候因子是5月(或说春季)降水和6月温度。树轮指数与各月PDSI相关值都很高(其中与当年5月的PDSI相关值高达0.614),都超过95%的置信水平,说明水热组合是该区域油松生长的主要限制因子。研究表明,影响本区油松生长的气候因子与我国北方地区油松生长的影响因子有较高的一致性,尤其是生长季前期的温度影响都很大;而其降水的影响及其与PDSI的相关都明显超前于黄土高原及北方地区,这既说明影响油松生长的大气候因子相似,又表明夏季风从东南向西北的不断推进。