Somatic embryogenesis and plantlet regeneration in Cornus florida

Somatic embryos were initiated from 12 to 15 weeks postanthesis (WPA) zygotic embryos of Cornus florida L. (flowering dogwood) cultured on Murashige-Skoog (MS) or Schenk and Hildebrandt (SH) medium amended with either 3 mg/L 2,4-D or 5 mg/L 2,4-D and 1 mg/L kinetin. White, opaque globular and early cotyledonary stage embryos were formed directly on detached cotyledons from 2 of the 5 trees sampled after 7 weeks of culture. Morphologically mature embryos developed after an additional 4 weeks incubation on medium without growth regulators; however, many of the embryos were fused in pairs along the entire length of the hypocotyl-radicle axis. Indirect embryogenesis was observed from callus cultures initiated from 9 to 15 WPA zygotic embryos. These cultures have continued to produce embryos for 16 months. Many of the embryos formed roots on germination medium, but only 12% formed plantlets and none developed past the first true leaf stage.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - FPA Formalin-propionic acid-ethanol (50%) - WPA weeks post-anthesis     

Anthocyanins in Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida fruits with health benefits

The anthocyanins in native Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida were quantified by HPLC and characterized by spectroscopic methods. The analyses of C. alternifolia and C. controversa revealed that both contained , and , respectively. Similarly, C. florida and C. kousa showed identical anthocyanin profiles with major anthocyanins as and cyanidin 3-O-glucoside (6), respectively. The amount of anthocyanins , and in C. alternifolia and C. controversa were 8.21, 8.44 and 0.02 mg; and 7.74, 5.92, and 0.02 mg/g of fresh fruits, respectively. The anthocyanins and in C. kousa and C. florida were 0.02 and 0.16 mg; and 0.62 and 0.03 mg/g fresh fruits, respectively. Anthocyanins and were not studied earlier for their inhibition of lipid peroxidation, cyclooxygenase enzymes (COX-1 and COX-2), and tumor cell proliferation. At 50 microg/mL, anthocyanins and inhibited lipid peroxidation by 71% and 68%, respectively. Similarly, they inhibited COX-1 enzymes by 39% and 49% and COX-2 enzyme by 54% and 48%, respectively, at 100 microg/mL. Anthocyanin displayed 50% growth inhibition (IC(50)) at 21, 25, 50, 60, and 75 microg/mL, against HCT-116 (colon), MCF-7 (breast), NCI-H460 (lung), SF-268 (central nervous system CNS), and AGS (stomach) human tumor cell lines, respectively. Similarly, IC(50) values for anthocyanin were 38, 30, 76, 100, and 100 microg/mL against HCT-116, MCF-7, NCI H460, SF-268, and AGS, respectively. This is the first report of the quantification and biological activities of anthocyanins in C. alternifolia, C. kousa and C. florida in addition to the anthocyanins not previously quantified in C. controversa.     

Foliar nutrient dynamics in tidal and non-tidal freshwater forested wetlands

Foliar nutrient dynamics were studied at 8 forest stands (three non-tidal and five tidal) along the lower 30 km of the Apalachicola River system in Florida, USA, during 2008. At each site, we sampled canopy foliage and litterfall from three to four trees representative of the dominant overstory species. Foliage and litterfall were analyzed for a variety of elements including N and P and these data were used to examine differences in element concentrations, nutrient ratios (C:P, C:N, and N:P), nutrient use efficiency, and nutrient proficiency. Measurements of tree diameter at basal height (DBH) at each plot were used with species allometric equations to estimate forest litterfall dry weight and N and P flux in non-tidal and tidal wetlands. Both non-tidal and tidal wetlands showed evidence of P limitation based on N:P ratios, but absolute levels of P were determined to be extremely low in tidal wetlands based on higher nutrient use efficiencies (measures of both P resorption efficiency and proficiency). Differences in P concentrations and fluxes between tidal and non-tidal wetlands are probably related to longer inundation and hydrologic export observed in tidal wetlands. Using estimations of annual litterfall dry weight and nutrient concentrations, N and P flux in non-tidal swamps were 2-4 times greater than in tidal wetlands. This study demonstrates the change in nutrient dynamics as wetlands shift from tidal to non-tidal conditions.     

Root membrane thermostability of Cornus florida L. provenances

Flowering dogwood seed collected from native trees in 3 USDA Hardiness Zones (6b, 7a, and 8a; American Horticultural Society (AHS) Heat-Zones 7, 7, and 8, respectively) were germinated and grown in containers prior to laboratory procedures to determine root cell membrane thermostability. Unsuberized, current season, fine root tissues were subjected to temperatures ranging from 20 to 60°C for 30 min and analyzed for cellular electrolyte leakage. Electrolyte leakage from root tissue exhibited a sigmoidal response to temperature for trees from each location. Critical midpoint temperature (T_m) was greater for seedlings native to USDA Hardiness Zone 6b (AHS Heat-Zone 7), 52.4±0.6°C, than T_m for seedlings originating from USDA Zone 7a (AHS Zone 7), 51.2±0.5°C. However, seedlings from USDA Zone 8a (AHS Zone 8), at 51.5±0.4°C, were similar to those collected in USDA Zones 6b (AHS Zone7) and 7a (AHS Zone 7). The results of this study find little genetic variability across this part of the native range of flowering dogwood regarding root thermotolerance. Although differences between two provenances were found, these differences may be of little ecological significance.     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

Freezing behaviours in wintering Cornus florida flower bud tissues revisited using MRI

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non‐invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high‐resolution magnetic resonance imaging (MRI). This allowed tissue‐specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to ?14 to ?21 °C or lower. The signal from other floral tissues decreased during cooling to ?7 °C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all‐or‐nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube‐based assay corroborated which tissues primarily froze. MRI is a powerful tool for non‐invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.     

Plant abiotic stress response and nutrient use efficiency

Abiotic stresses and soil nutrient limitations are major environmental conditions that reduce plant growth, productivity and quality.Plants have evolved mechanisms to perceive these environmental challenges, transmit the stress signals within cells as well as between cells and tissues, and make appropriate adjustments in their growth and development in order to survive and reproduce. In recent years, significant progress has been made on many fronts of the stress signaling research, particularly in understanding the downstream signaling events that culminate at the activation of stress-and nutrient limitation-responsive genes, cellular ion homeostasis, and growth adjustment. However, the revelation of the early events of stress signaling, particularly the identification of primary stress sensors, still lags behind. In this review, we summarize recent work on the genetic and molecular mechanisms of plant abiotic stress and nutrient limitation sensing and signaling and discuss new directions for future studies.     

Dinucleotide microsatellite loci isolated from flowering dogwood (Cornus florida L.)

We report eight variable dinucleotide microsatellite loci cloned from flowering dogwood (Cornus florida L.) using a biotin enrichment protocol. Degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR) was used to generate a population of DNA fragments, from which adenine‐cytosine dinucleotide (AC) and adenine‐guanine dinucleotide (AG) repeats were captured using biotinylated probes and streptavidin coated magnetic particles. The captured fragments were cloned into plasmids, and the plasmid library was screened for microsatellites using a simple PCR technique. Selected plasmids were sequenced, and PCR primers were designed and optimized using a thermal‐gradient thermocycler. The loci reported are highly variable with an average of 9.25 allele per locus and an average heterozygosity of 0.84.     

Enhanced plant nutrient use efficiency with PGPR and AMF in an integrated nutrient management system

A 3 year field study was conducted with field corn from 2005 to 2007 to test the hypothesis that microbial inoculants that increase plant growth and yield can enhance nutrient uptake, and thereby remove more nutrients, especially N, P, and K from the field as part of an integrated nutrient management system. The field trial evaluated microbial inoculants, which include a commercially available plant growth-promoting rhizobacteria (PGPR), arbuscular mycorrhiza fungi (AMF), and their combination across 2 tillage systems (no-till and conventional till) and 2 fertilization regimes (poultry litter and ammonium nitrate). Data were collected on plant height, yield (dry mass of ears and silage), and nutrient content of corn grain and silage. In addition, nutrient content of soil was determined, and bioavailability of soil nutrient was measured with plant root simulator probes. Results showed that inoculants promoted plant growth and yield. For example, grain yields (kg.ha(-1)) in 2007 for inoculants were 7717 for AMF, 7260 for PGPR+AMF, 7313 for PGPR, 5725 for the control group, and for fertilizer were 7470 for poultry litter and 6537 for NH4NO3. Nitrogen content per gram of grain tissues was significantly enhanced in 2006 by inoculant, fertilizer, and their interactions. Significantly higher amounts of N, P, and K were removed from the plots with inoculants, based on total nutrient content of grain per plot. These results supported the overall hypothesis and indicate that application of inoculants can lead to reduction in the build up of N, P, and K in agricultural soils. Further studies should be conducted to combine microbial inoculants with reduced rates of fertilizer.     

Ecological genomics of local adaptation in Cornus florida L. by genotyping by sequencing

Discovering local adaptation, its genetic underpinnings, and environmental drivers is important for conserving forest species. Ecological genomic approaches coupled with next‐generation sequencing are useful means to detect local adaptation and uncover its underlying genetic basis in nonmodel species. We report results from a study on flowering dogwood trees (Cornus florida L.) using genotyping by sequencing (GBS). This species is ecologically important to eastern US forests but is severely threatened by fungal diseases. We analyzed subpopulations in divergent ecological habitats within North Carolina to uncover loci under local selection and associated with environmental–functional traits or disease infection. At this scale, we tested the effect of incorporating additional sequencing before scaling for a broader examination of the entire range. To test for biases of GBS, we sequenced two similarly sampled libraries independently from six populations of three ecological habitats. We obtained environmental–functional traits for each subpopulation to identify associations with genotypes via latent factor mixed modeling (LFMM) and gradient forests analysis. To test whether heterogeneity of abiotic pressures resulted in genetic differentiation indicative of local adaptation, we evaluated F_st per locus while accounting for genetic differentiation between coastal subpopulations and Piedmont‐Mountain subpopulations. Of the 54 candidate loci with sufficient evidence of being under selection among both libraries, 28–39 were Arlequin–BayeScan F_st outliers. For LFMM, 45 candidates were associated with climate (of 54), 30 were associated with soil properties, and four were associated with plant health. Reanalysis of combined libraries showed that 42 candidate loci still showed evidence of being under selection. We conclude environment‐driven selection on specific loci has resulted in local adaptation in response to potassium deficiencies, temperature, precipitation, and (to a marginal extent) disease. High allele turnover along ecological gradients further supports the adaptive significance of loci speculated to be under selection.     

中国东部南北样带森林优势植物叶片的水分利用效率和氮素利用效率

通过测定中国东部南北样带主要森林生态系统中10种优势植物(兴安落叶松、蒙古栎、水曲柳、紫椴、色木槭、红松、杉木、木荷、马尾松、锥栗)叶片的碳氮含量(Cmass、Nmass)、同位素丰度(δ13C、δ15N)以及光合响应曲线,分析了不同优势植物叶片的水分利用效率和氮素利用效率之间的差异及其相互关系.结果表明： 不同生活型植物叶片的Nmass和δ15N差异显著,表现为阔叶植物>针叶植物,落叶植物>常绿植物;最大光合速率(Pn max)表现为针叶植物>阔叶植物,落叶植物>常绿植物;植物叶片的瞬时水分利用效率(WUEi)和长期水分利用效率(WUE)均表现为阔叶植物>针叶植物,常绿植物>落叶植物;植物叶片的瞬时氮素利用效率(NUEi)和长期氮素利用效率(NUE)则表现出相反的规律,且常绿植物和落叶植物叶片的NUE差异显著;WUEi和WUE之间相关性不显著,而NUEi和NUE之间呈显著正相关.植物叶片的水分利用效率与氮素利用效率显著负相关.两种资源利用效率均受植物生活型的影响,并且存在一定的制约关系.     

亚热带红壤侵蚀区马尾松针叶养分含量及再吸收特征

叶片衰老过程中的养分再吸收是植物适应养分贫瘠生境的一种重要策略,一直是生态学领域的研究热点。以亚热带红壤侵蚀区生态恢复先锋树种马尾松为研究对象,分析4种不同恢复水平下马尾松叶片养分含量随叶龄的变化情况及养分再吸收特征。结果表明:(1)叶片中N、P、K含量变化范围分别在(6.10±0.52)—(12.02±0.85)mg/g、(0.17±0.03)—(1.02±0.01)mg/g、(1.58±0.49)—(9.46±0.90)mg/g,随叶龄增长整体呈先增加后降低的趋势,具有一个快速积累期和一个相对漫长衰减期的动态特征,这表明叶龄也是影响叶片养分含量的重要因素;(2)叶片N、P、K含量随生境恢复水平的提高而增加,且N、P含量在除凋落叶外的叶龄阶段均表现出显著正相关,表明叶片中这两种营养元素在动态变化上存在协同性;(3)在叶片N、P、K再吸收效率中,P、K再吸收效率较高,而N相对较低。养分再吸收受生境中营养元素的含量水平、循环方式等因素的综合影响表现出一定选择性,对生境中较为贫瘠或使其生长受到限制的元素具有较高的再吸收效率。这不仅可以减小植物对外源养分的依赖性,同时也维持了体内重要营养元素的平衡。随叶龄增长叶片中N/P逐渐增大,反映出在叶片衰老过程中对限制元素P具有较强的再吸收能力,这种反馈调节提高了马尾松对养分贫瘠环境的适应性。本文的研究结果可为亚热带红壤侵蚀退化区先锋物种在贫瘠生境条件下的养分利用机制与适应对策方面的研究提供理论依据。     

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

中亚热带森林转换对凋落物养分归还及养分利用效率的影响

为了解中亚热带森林转换对森林生态系统碳及养分循环的影响,以中亚热带米槠天然林、森林转换后的米槠次生林和杉木人工林为对象,对3种林分的凋落物量、养分归还量和养分利用效率进行4年研究.结果 表明:米槠天然林转换为米槠次生林和杉木人工林后,年凋落物量分别下降29.0％和45.7％,凋落物氮归还量分别下降34.0％和72.7％...     

In vitro detection of Cornus florida callus insensitive to toxic metabolites of Discula destructiva

Summary Dogwood anthracnose, caused by the fungus Discula destructiva Redlin, is a severe disease of flowering dogwood (Cornus florida L.) and Pacific dogwood (C. nuttallii Aud.). Disease control is inadequate in nurseries and landscapes and absent in the forest, and resistant cultivars are not commercially available. The ability to select tissues insensitive to culture filtrates from D. destructiva in vitro offers a novel and important approach for the selection of dogwood genotypes that are resistant to or tolerant of this devastating fungus. Embryo-derived dogwood callus cultures were established on Murashige and Skoog medium amended with benzyladenine (BA) and either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA). Selection for insensitivity to D. destructiva metabolites was done by placement of individual cultures on media amended with progressively higher concentrations of a partially purified culture filtrate (PPCF) containing lowmolecular-weight compounds. Following this selection process, cultures were challenged in a dose-response format with PPCF to determine whether the sensitivity of the callus to the culture filtrate had changed. During the selection period, the fresh weight of callus grown on medium containing 2,4-D and amended with PPCF was always less than that of callus grown on medium amended with the same concentration of potato-dextrose broth (PDB, negative control). Fresh weight of callus was greater on medium containing NAA amended with PPCF than on medium with the same concentration of PDB. Callus selected in the presence of NAA showed decreased sensitivity to toxic metabolites at higher concentrations of culture filtrate. The in vitro system described may assist in the identification of disease-resistant germplasm important to the long-term survival of flowering dogwood.     

Discovering variation of secondary metabolite diversity and its relationship with disease resistance in Cornus florida L.

Understanding intraspecific relationships between genetic and functional diversity is a major goal in the field of evolutionary biology and is important for conserving biodiversity. Linking intraspecific molecular patterns of plants to ecological pressures and trait variation remains difficult due to environment‐driven plasticity. Next‐generation sequencing, untargeted liquid chromatography–mass spectrometry (LC‐MS) profiling, and interdisciplinary approaches integrating population genomics, metabolomics, and community ecology permit novel strategies to tackle this problem. We analyzed six natural populations of the disease‐threatened Cornus florida L. from distinct ecological regions using genotype‐by‐sequencing markers and LC‐MS‐based untargeted metabolite profiling. We tested the hypothesis that higher genetic diversity in C. florida yielded higher chemical diversity and less disease susceptibility (screening hypothesis), and we also determined whether genetically similar subpopulations were similar in chemical composition. Most importantly, we identified metabolites that were associated with candidate loci or were predictive biomarkers of healthy or diseased plants after controlling for environment. Subpopulation clustering patterns based on genetic or chemical distances were largely congruent. While differences in genetic diversity were small among subpopulations, we did observe notable similarities in patterns between subpopulation averages of rarefied‐allelic and chemical richness. More specifically, we found that the most abundant compound of a correlated group of putative terpenoid glycosides and derivatives was correlated with tree health when considering chemodiversity. Random forest biomarker and genomewide association tests suggested that this putative iridoid glucoside and other closely associated chemical features were correlated to SNPs under selection.     

Properties of Internal Water Exchange in Leaves of Ilex opaca Ait. and Cornus florida L.

Resistances, capacitances, and time constants for internal watertransfer were estimated in leaves from naturally-occurring individualsof Ilex opaca Ait. and Cornus florida L., both common under-storytree species in the North Carolina piedmont deciduous forest.The water exchange properties depended strongly on the currentwater status of the leaf tissue. Resistance to water transferfrom leaf storage sites, capacitance of storage sites, and thetime constant for transfer of water out of storage sites allincreased as tissue water deficits developed. The exchange propertieswere determined over a range of leaf water potentials whichthe plants typically experienced in the field. The measuredresponses are viewed as properties integrally associated withother aspects of water transfer elsewhere in the plant and inthe soil and atmosphere, and may be significant in determiningleaf water deficits under a fluctuating environment.     

植物养分利用效率研究综述

养分利用效率的概念是理解生态系统功能的中心。本文从植物养分利用效率的概念出发,对养分利用效率的表示与计算方法、影响因素以及养分再吸收的生物化学基础等进行综述,分析目前研究中存在的问题,最后指出今后应加强研究的方面。     

Foliar nutrient concentrations of small Chamaecyparis in Taiwan

Summary Seedlings ofChamaecyparis lawsoniana, C. formosensis andC. taiwanensis were grown in a forest nursery in Taiwan. Native saplings of the latter two species were sampled in a clearcut and in the forest understory. Foliar nutrient concentrations were mostly in the mid-range of values for other conifers. However, Ca (.59–1.5%) and Cu (7–20 ppm) were almost always high. Concentrations of N, P, and K in field saplings in Taiwan were low enough (.7–1.5%, .07–.15%, .47–.60%, respectively) to indicate that these elements are probably limiting growth.Among the three species,C. lawsoniana usually had the lowest foliar nutrient values. When a difference among the species from Taiwan occurred,C. taiwanensis usually was lower. Foliar nutrient concentration varied among provenances, especially withinC. formosensis. Seasonal variability did not occur consistently; nutrients which did change increased their concentrations from the dormant to the growing season, except for K which decreased.     

On the relationship between nutrient use efficiency and fertility in forest ecosystems

The concept of nutrient use efficiency is central in understanding ecosystem functioning because it is the step in which plants can influence the return of the nutrients to the soil pool and the quality of the litter. There are several ways to define nutrient use efficiency, but a common way within ecosystem ecology is as the ratio of litterfall production per unit nutrient to the litterfall nutrient content. However, this ratio is not a valid measurement to examine nutrient use efficiency in relationship to ecosystem fertility because there is a strong autocorrelation between litterfall dry mass per unit of nutrient and the amount of nutrients. More appropriate statistical analysis of the relationship between the fertility of ecosystems and the amount of nutrients in the litterfall are inconclusive, but indicate that, at least in some cases, there is (1) no pattern, (2) higher nutrient use efficiency at intermediate-fertility sites or (3) higher efficiency at higher-fertility sites. There is, however, no indication that nutrient use efficiency is greater in nutrient-poor ecosystems. This conclusion has important consequences for ecosystem nutrient cycling. Given the lack of a clear, consistent relationship between site fertility and litterfall nutrients, there is little likelihood that such a feedback mechanism plays an important role in ecosystem nutrient cycling. Received: 22 January 1996 / Accepted: 26 December 1996