Changes in antioxidant enzyme activity in the fine roots of black poplar (Populus nigra L.) and cottonwood (Populus deltoides Bartr. ex Marsch) in a heavy-metal-polluted environment

The effect of heavy metal deposition onto soil from a copper smelter on lipid peroxidation and antioxidant enzyme activity in the fine roots of two poplars (Populus nigra L. and Populus deltoides Bartr. ex Marsch) was analyzed. The subjects were mature trees growing in real environments. In both analyzed species, heavy metals stimulated the overproduction of free radicals in fine roots (measured as malondialdehyde level), which was directly proportional to advancing senescence. In young fine roots, heavy metals caused a decrease in guaiacol peroxidase activity and presumably disturbed the lignification process. Catalase was highly sensitive to the presence of heavy metals in the soil. In contrast, ascorbate peroxidase and glutathione reductase activities were unaffected by heavy metals. In the case of superoxide dismutase, a clear increase in enzyme activity was observed only in P. nigra under drought conditions, whereas it was inhibited in polluted stands.     

Pollen competition among Populus deltoides Marsh., P. nigra L. and P. maximowiczii Henry in fertilizing P. deltoides ovules and siring its seed crop

Summary Interspecific pollen competition among Populus deltoides, P. nigra and P. maximowiczii in fertilizing P. deltoides ovules was studied by using a pollen mixture technique, allozymes and leaf morphology. The frequencies of F₁ seedlings of different paternities in pollen-mix crosses showed highly significant (P<0.01) departures from the 11 ratio expected if pollen selection was random. P. deltoides pollen was the most competitive. The mean percentages of F₁ seedlings of P. deltoides paternity in crosses with pollen mixes P. deltodes + P. nigra, P. deltoides + P. maximowiczii, and P. deltoides + P. nigra + P. maximowiczii were 95.0, 92.5, and 84.8, respectively. P. maximowiczii pollen was more competitive than P. nigra pollen, which was at a selective disadvantage. An average of 83.6% of F₁ progenies of the eight crosses with P. nigra + P. maximowiczii pollen showed P. maximowiczii paternity. Also, in four crosses with P. deltoides + P. nigra + P. maximowiczii pollen, the relative proportion of P. deltoides × P. maximowiczii seedlings (13.4%) was higher than that of P. deltoides × P. nigra seedlings (1.8%). Pollen proportions in the pollen mixes and pollen size did not significantly affect the competitive ability of the pollen. The relative pollen competitive ability indicated reproductive affinities among the species.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

Variations in levels of clonality among Populus nigra L. stands of different ages

The evolution of genotypic diversity with population age remains poorly explored in clonal plant populations despite the potential for important shifts to occur through the course of time. Woody sprouting species are particularly under-represented in studies investigating intra-specific variations in levels of clonality from one locality to the next and through time. In this study we sought to determine the incidence and frequency of replicate genotypes in natural Populus nigra L. (Salicaceae) stands of different ages. Ten stands of this woody riparian sprouting species were selected in each of three distinct age groups (young, middle-aged and old) along a 30 km stretch of the River Garonne (south-west France). Leaf samples were collected from 15 neighbouring trees in each stand (450 samples in total) and replicate genotypes were identified using five SSR markers. Replicate genotypes were identified in two-thirds of all stands sampled (i.e. 50 of young stands, 100 of middle-aged stands and 50% of old stands). Young stands had significantly fewer replicated genotypes than middle-aged or old stands, while middle-aged stands had the greatest number of replicated genotypes. Replicate genotypes were most often found to occur as nearest neighbours and formed relatively small, discrete units (i.e. 2–4 trees growing in close proximity to one another). This suggests that asexual regeneration frequently occurs through flood-training in this species, although asexual regeneration from translocated fragments also evidently occurs as evidenced by 11 cases of replicate genotypes occurring in widely separated stands (up to 19 km apart). The results of this study highlight the need for a hierarchical sampling strategy in space and across age groups for an accureate understanding of the genotypic structure of woody sprouting species populations. Conservation and management of effective population sizes will benefit from better insight into not only spatial, but also temporal variations in levels of genotypic diversity.Co-ordinating editor: J. Tuomi     

A large scale survey of Populus nigra presence and genetic introgression from non-native poplars in Switzerland based on molecular identification

Populus nigra is considered a rare and threatened tree species in Switzerland because of dramatic habitat loss owing to river regulations during the last two centuries and because of potential gene introgression from non-indigenous P. deltoides through planted P. x canadensis hybrids. The significance of introgression as an endangerment to P. nigra, however, is controversial. The aims of the present study were (1) to assess how abundant P. nigra trees are in Switzerland and (2) to assess potential gene introgression. We present data from a molecular survey of 1372 putative P. nigra trees from Switzerland, using both chloroplast DNA and nuclear DNA markers. The results show that P. nigra is more abundant in Switzerland than hitherto thought. Furthermore, we detected a low frequency of gene introgression.     

Pathogenicity of Beauveria bassiana (Deuteromycotina: Hypomycetes) to Larvae of the Small Poplar Longhorn Beetle, Saperda populnea (Coleoptera: Cerambycidae)

The small poplar longhorn beetle, Saperda populnea is an important pest of Lombardy poplars (Populus nigra L.) in Turkey. A survey for natural entomopathogenic fungi of S. populnea larvae was made in Erzurum, Turkey, during the period 2004–2005. Larvae (13.5%) infected with a strain of the fungus Beauveria bassiana were found. The pathogenicity of B. bassiana strain 46 was conducted with different concentrations of conidia (10⁶, 10⁷ and 10⁸ conidia/ml) of this isolate on S. populnea larvae. The lowest concentration (10⁶ conidia/ml) caused about 56% mortality within 6 days. One hundred percent mortality was achieved after median lethal time (LT₅₀) of 4.6 and 4.4 days for 10⁷ and 10⁸ conidia/ml, respectively. There were no significant differences between median lethal times. This is the first record of natural infection of S. populnea larvae by B. bassiana.     

Identification and characterization of CYP79D6v4, a cytochrome P450 enzyme producing aldoximes in black poplar (Populus nigra)

After herbivore feeding, poplar trees produce complex volatile blends containing terpenes, green leaf volatiles, aromatics, and nitrogen-containing compounds such as aldoximes and nitriles. It has been shown recently that volatile aldoximes released from gypsy moth (Lymantria dispar) caterpillar-damaged black poplar (Populus nigra) trees attract parasitoids that are caterpillar enemies. In western balsam poplar (P. trichocarpa), volatile aldoximes are produced by 2 P450 monooxygenases, CYP79D6v3 and CYP79D7v2. A gene fragment with high similarity to CYP79D6/7 was recently shown to be upregulated in herbivore-damaged leaves of P. nigra. In the present study we report the cloning and characterization of this gene, designated as CYP79D6v4. Recombinant CYP79D6v4 was able to convert different amino acids into the corresponding aldoximes, which were also found in the volatile blend of P. nigra. Thus, CYP79D6v4 is most likely involved in herbivore-induced aldoxime formation in black poplar.     

小叶杨Δ~1-吡咯琳-5-羧酸合成酶(P5CS)基因克隆及在杂种落叶松中的转化

以小叶杨为材料构建了干旱胁迫和正常生长条件下的cDNA文库,以特异性引物从中扩增出一条1 850bp大小的DNA片段,经序列分析证实该片段编码Δ1-吡咯琳-5-羧酸合成酶(P5CS)。将该片段构建入植物表达载体pBI121中,在落叶松杂交育种中利用花粉管通道法将带有该P5CS基因的植物表达质粒转化入杂种落叶松,收获球果取出种子,提取转化种子发芽长出幼芽的DNA,特异性PCR扩增和Southern,Western Blotting检测证实落叶松中已导入P5CS基因。     

Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. ex Marsh.

Microautoradiography was used to follow the translocation pathways of ¹⁴C-labeled photosynthate from mature source leaves, through the stem, to immature sink leaves three nodes above. Translocation occurred in specific bundles of the midveins and petioles of both the source and sink leaves and in the interjacent internodes. When each of six major veins in the lamina of an exporting leaf was independently spot-fed ¹⁴CO₂, label was exported through specific bundles in the petiole associated with that vein. When the whole lamina of a mature source leaf was fed ¹⁴CO₂, export occurred through all bundles of the lamina, but acropetal export in the stem was confined to bundles serving certain immature sink leaves. Cross-transfer occurred within the stem via phloem bridges. Leaves approaching maturity translocated photosynthate bidirectionally in adjacent subsidiary bundles of the petiole. That is, petiolar bundles serving the lamina apex were exporting unlabeled photosynthate while those serving the lamina base were simultaneously importing labeled photosynthate. The petioles and midveins of maturing leaves were strong sinks for photosynthate, which was diverted from the export front to differentiating structural tissues. The data support the idea of bidirectional transport in adjacent bundles of the petiole and possibly in adjacent sieve tubes within an individual bundle.Abbreviations C central leaf trace - L left leaf trace - LPI leaf plastochron index - R right leaf trace     

Phyllotactic transitions in the vascular system of Populus deltoides bartr. as determined by 14C labeling

Populus deltoides seedlings progress through 2/5, 3/8, and 5/13 orders of phyllotaxis in attaining Plastochron Index 16 (PI 16). The manner in which the vascular system was reoriented during these phyllotactic transitions was determined by anatomical analysis of serial microsections, whereas the positions of the transitions were determined by ¹⁴C labeling. The midvein at the tip of leaves representing plants of different PI and leaves of different Leaf Plastochron Index (LPI) was fed ¹⁴CO₂ photosynthetically, and primordia LPI 0 through LPI-9 were dissected from the buds and analyzed for ¹⁴C. By combining the labeling data with the anatomical observations it was possible to reconstruct the vascular system of a plant of PI 16 and to locate the phyllotactic transitions. Both the anatomical and the labeling data showed a high degree of reproducibility among plants suggesting that the phyllotactic pattern to which the vascular system conforms may be programmed in the plant and transmitted acropetally through the developing leaves and procambial strands. The origin of new primordia and the concepts of orthostichy, ontogenetic helix, and Fibonacci sequence are discussed as they apply to the vascular system of P. deltoides.     

Inhibition of cinnamyl-alcohol-dehydrogenase activity and lignin synthesis in poplar (Populus x euramericana Dode) tissues by two organic compounds

Two organic compounds, N-(O-hydroxyphenul)-and N-(O-aminophenyl)sulfinamoyltertiobutyl acetate (OHPAS and NH₂PAS, respectively) have been designed for inhibiting cinnamylalcohol dehydrogenase (EC 1.1.1.2), a zinc metalloenzyme involved specifically in lignification. This paper describes their effects in vitro on the activity of the enzyme isolated from poplar and in vivo on the lignification of poplar tissues. The enzyme inhibition was time- and dose-dependent and pseudoirreversible indicating that these compounds could act as suicide inhibitors. Neither OHPAS nor NH₂PAS exhibited affinity towards other plant zinc metalloenzymes or phenolic enzymes tested. Only NH₂PAS exerted an effect on cinnamoyl: CoA reductase, another specific enzyme of lignification. In addition, these inhibitors, at the concentration of 80 M, reduced the fluxes of lignin synthesis in poplar stems by 45%. These results show that OHPAS and NH₂PAS could become useful tools for reducing lignification in plants.Abbreviations CDH cinnamyl alcohol dehydrogenase - NH₂PAS N (O-aminophenyl) sulfinamoyltertiobutyl acetate - OHPAS N(O-hydroxyphenyl) sulfinamoyltertiobutyl acetate     

Male Social Behavior and Dominance Hierarchy in the Sulawesi Crested Black Macaque (Macaca nigra)

In a 6-week study of the social behavior of wild Sulawesi crested black macaques (Macaca nigra), we found a linear and transitive dominance hierarchy among the six adult males in one social group. Dominance rank, as determined by the direction of supplantations, correlated strongly with percentage of time near more than four neighbors, frequency of grooming received from adult females, and percentage of time with an adult female as nearest neighbor. These results suggest that high-ranking males are socially attractive. Adult females sexually solicited high-ranking males more often than low-ranking males, but frequency of copulation was not correlated with dominance rank. Frequency and intensity of aggression between males are strongly correlated with rank distance, but aggression toward females was greatest for mid-ranking males. Males of all rank displayed significantly more aggression toward sexually receptive females than toward females in other estrous states. These data indicate that male Sulawesi crested black macaques display a social organization similar to that reported for multimale groups in other macaque species rather than the egalitarian social organization described for female Sulawesi macaques.     

Behavior, Diet, and Movements of the Sulawesi Crested Black Macaque (Macaca nigra)

We present the first field study of activity budgets, diet and ranging patterns of the Sulawesi crested black macaque, Macaca nigra, one of seven macaque species endemic to Sulawesi, Indonesia. We studied three crested macaque groups, ranging in size from 50 to 97 individuals, for 18 months in the Tangkoko-DuaSudara Nature Reserve, North Sulawesi. They spent 59% of the day moving and procuring food, especially fruits, and 41% of the day resting and socializing. Their diet is composed of more than 145 species of fruit (66% of observed feeding bouts), vegetative material (2.5%), invertebrates (31.5%), and occasional vertebrate prey. Group differences were more pronounced than seasonal or diurnal differences. Specifically, the largest group moved farther during the day, moved at a faster and more uniform rate, ate less fruit, rested more, and socialized less than the smaller groups did. The largest group had the largest home range, but it included less primary forest and more disturbed habitat than the ranges of smaller groups. There are individual differences in activity budgets of adult males and females in time spent moving, resting, feeding, and socializing that may reflect differences in reproductive strategies of males versus females. The behavior of large juveniles is more similar to that of adults than to that of small juveniles. Daily movements and use of home range are correlated with diet. Macaques moved shorter distances as the proportion of time spent feeding on fruit increased, and the top four dietary items accounted for most of the variance in entry into hectare blocks of home range.     

Molecular status of the dusky Canada goose (Branta canadensis occidentalis): A genetic assessment of a translocation effort

Until recently, the dusky Canada goose (Branta canadensis occidentalis) was managedas one breeding population from the CopperRiver Delta (CRD), Alaska. Population numberson the CRD have declined precipitously over thelast three decades, due in part to changes inhabitat. In 1981, a pair of Canada geese,presumably B.c. occidentalis, wasreported nesting on Middleton Island (MID), inthe Gulf of Alaska. Numbers of Canada geese onthe island increased in the decade subsequentto a translocation of geese from CRD to MID,but it is unclear whether the increase isattributable to the translocation effort. Weused genetic data derived from three classes ofgenetic markers to clarify relationships ofCanada geese breeding in south-coastal Alaska. Geese were sampled from 5 populations: CRD,MID, Anchorage (ANC), Admiralty Island (ADM) insoutheastern Alaska, and Green Island (GRN) inPrince William Sound (PWS). Mitochondrial DNAanalyses demonstrate Canada geese from MID arenearly monomorphic for a unique haplotype fixedon GRN but not found in CRD or any otherbreeding population. Furthermore, nuclearmarkers consistently cluster MID with GRN tothe exclusion of CRD. We suggest the currentpopulation on MID is not derived from birdstranslocated from CRD, but rather that MID wasmost likely colonised by birds inhabiting otherisland habitats within the PWS. Furthermore,since geese from the CRD share mtDNA haplotypeswith geese from other breeding locales, theyapparently share recent common ancestry and/orgene flow with populations representing othersubspecies. Our genetic data raise questionsabout the validity of current management unitsof Canada geese.     

Spatial and temporal distribution of the alkaloid sanguinarine in Sanguinaria canadensis L. (Bloodroot)

Acclimated and non-acclimated potted plants of Sanguinaria canadensis L. were harvested at early and late dormancy, anthesis, and immature and mature fruiting stages. Sanguinarine content and concentration were determined for rhizomes (distal, proximal, and middle sections), roots, leaves, flower, and fruit. Rhizomes had highest sanguinarine content and concentrations, and exhibited decreasing concentration gradients from the distal to proximal third. Concentrations in roots were a tenth of rhizome concentration. Concentrations in leaves, flowers, and fruit were one-thousandth of rhizome Sanguinarine content in whole acclimated plants was constant. Content in whole nonacclimated plants increased as the plant became physiologically active, but was constant during fruit maturation: content in roots, leaves, and fruit did not change. The substantial increase in whole-plant dry weight coupled with the unchanging sanguinarine content during fruit maturation suggests either a shift in photosynthate allocation from defense to growth, or a constant turnover of sanguinarine.     

Genetic variation of stomatal traits and carbon isotope discrimination in two hybrid poplar families (Populus deltoides 'S9-2' x P. nigra 'Ghoy' and P. deltoides 'S9-2' x P. trichocarpa 'V24')

Background and Aims

Stomata play an important role in both the CO₂ assimilation and water relations of trees. Therefore, stomatal traits have been suggested as criteria for selection of clones or genotypes which are more productive and have larger water-use efficiency (WUE) than others. However, the relationships between plant growth, WUE and stomatal traits are still unclear depending on plant material (genus, species, families, genotypes) and, more precisely, on the strength of the relationships between the plants. In this study, the correlations between these three traits categories, i.e. plant growth, WUE and stomatal traits, were compared in two related poplar families.

Methods

Stomatal traits (stomatal density, length and ratio adaxial : abaxial stomatal densities) of a selection of F₁ genotypes and the parents of two hybrid poplar families Populus deltoides ‘S9-2’ × P. nigra ‘Ghoy’ (D × N family, 50 F₁) and P. deltoides ‘S9-2’ × P. trichocarpa ‘V24’ (D × T family, 50 F₁) were measured, together with stem height and circumference. Carbon isotope discrimination (Δ) was determined and used as an indicator of leaf-level intrinsic WUE.

Key Results

Leaves of hybrids and parents were amphistomatous, except for the P. trichocarpa parent. Both families displayed high values of heritability for stomatal traits and Δ. In the progeny, the relationship between stem circumference and Δ was weak for the D × N family, while abaxial and total stomatal density were positively associated with stem dimensions for the D × T family only.

Conclusions

Genetic variation in stomatal traits and Δ was large within as well as between the different poplar species and their hybrids, but there were no direct relationships between stomatal traits and plant growth or Δ. As already noticed in various poplar hybrids, the absence of, or the weak, relationship between Δ and plant growth allows the possibility of selecting poplar genotypes combining high productivity and high WUE. In this study, stomatal traits are of limited value as criteria for selection of genotypes with good growth and large WUE.Key words: Adaxial and abaxial stomatal density, stomatal length, heritability, water-use efficiency (WUE), F₁ hybrids, breeding, Populus deltoides, Populus nigra, Populus trichocarpa     

A fast and simple genetic survey reveals the spread of poplar hybrids at a natural Elbe river site

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P.  × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.     

Regulation of sulfate uptake and xylem loading of poplar roots (Populus tremula x P. alba)

Sulfate transport processes and its regulation were studied in roots of poplar trees (Populus tremula x P. alba). From the exponential increase in sulfate uptake with temperature an activation energy (E_a) of 9.0±0.8 kJ mol^–1 was calculated. In the concentration range 0.005–10 mM sulfate uptake showed biphasic Michaelis-Menten kinetics with a K_m of 3.2±3.4 M and a V_max of 49±11 nmol SO₄^2– g^–1 FW h^–1 for the high-affinity uptake system (phase 1) and a K_m of 1.33±0.41 mM and a V_max of 255±25 nmol SO₄^2– g^–1 FW h^–1 for the low-affinity system (phase 2). Xylem loading decreased linearly with temperature and remained unchanged within the sulfate concentration range studied. Regulation of sulfate uptake and xylem loading by O-acetyl serine (OAS), Cys, reduced glutathione (GSH), Met and S-methylmethionine (SMM) were tested by perfusion into the xylem sap with the pressure probe and by addition to the incubation medium. When added directly to the transport medium, Cys and GSH repressed, and OAS stimulated sulfate uptake; xylem loading was stimulated by Cys, repressed by GSH and only slightly affected by OAS. When perfused into the xylem, none of the compounds tested affected sulfate uptake of excised roots, but xylem loading was stimulated by SMM and OAS and repressed by Met. Apparently, the site of application strongly determined the effect of regulatory compounds of sulfate transport processes.