Agrobacterium tumefaciens‐mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva

Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strategy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phosphotransferase (hph) flanked by upstream and downstream sequences of polyketide synthase‐like (PKS‐L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequencing and Southern blotting. No significant differences were observed in melanin production between PKS‐L1 disruptants and wild type isolates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study.     

Thigmomorphogenesis: changes in the morphology and mechanical properties of two Populus hybrids in response to mechanical perturbation

To identify hybrid-specific differences in developmental response to mechanical perturbation (MP), we compared the effects of stem flexure on several morphological and mechanical properties of two Populus trichocarpa Torr. & A. Gray x P. deltoides Bartr. ex Marsh. hybrids, 47-174 and 11-11. In response to the MP treatment, both hybrids exhibited a significant increase in radial growth, especially in the direction of the MP (47-174, P = 0.0001; 11-11, P = 0.002), and a significant decrease in height to diameter growth ratio (P = 0.0001 for both hybrids), suggesting that MP-treated stems are more tapered than control stems. A direct consequence of the MP-induced increase in radial growth was a significant increase in flexural rigidity (EI, N mm(2)) in stems of both hybrids (47-174, P = 0.0001; 11-11, P = 0.009). Both control and MP-treated stems of Hybrid 47-174 had significantly greater height to diameter ratios and EI values than the corresponding stems of Hybrid 11-11 (11-11 stem ratios and EI values were 85 and 76%, respectively, of those of 47-174). In Hybrid 47-174, Young's modulus of elasticity (E, N mm(-2)), a measure of stem flexibility, for MP-treated stems was only 80% of the control value (P = 0.0034), whereas MP had no significant effect on E of stems of Hybrid 11-11 (P = 0.2720). Differences in flexure response between the hybrids suggest that Hybrid 47-174 can produce a stem that is more tolerant of wind-induced flexure by altering both stem allometry and material properties, whereas Hybrid 11-11 relies solely on changes in stem allometry for enhanced stability under MP conditions.     

Effects of two iron sources on iron and cadmium allocation in poplar (Populus alba) plants exposed to cadmium

Effects of 10 microM cadmium (supplied as Cd nitrate) on the utilization and allocation of iron (Fe) were investigated in poplar (Populus alba L.) plants grown in nutrient solution with Fe(III)-EDTA or Fe(III)-citrate as the Fe source. The effects of Cd were also compared with those of Fe deprivation. The accumulation of Fe in roots was 10-fold higher in plants grown with Fe-citrate than with Fe-EDTA. Cadmium decreased leaf chlorophyll concentrations and photosynthetic rates, and these decreases were more marked in plants grown with Fe-citrate than with Fe-EDTA. In both Fe treatments, addition of Cd caused large increases in root and shoot apoplasmic and non-apoplasmic Cd contents and increases in root Fe content; however, Cd decreased shoot Fe content, especially in plants grown with Fe-citrate. New leaves of plants grown with Fe-citrate had small cellular (non-apoplasmic) Fe pools, whereas these pools were large in new leaves of plants grown with Fe-EDTA. Non-apoplasmic Cd pools in new leaves were smaller in plants grown with Fe-citrate than with Fe-EDTA, indicating that inactivation of non-apoplasmic Cd pools is facilitated more by Fe-EDTA than by Fe-citrate. In the presence of Cd, Fe-EDTA was also superior to Fe-citrate in maintaining an adequate Fe supply to poplar shoots. Differences in plant responses to Fe-EDTA and Fe-citrate may reflect differences in long-distance transport of Fe rather than in acquisition of Fe by roots.     

ELISA Quantitation and GC-MS identification of abscisic acid in stigma, ovary and pedicel of pollinated poplar flowers (Populus nigra L.)

A method of fractionation, quantitation and identification of abscisic acid (ABA) in poplar flowers is presented in the context of intra- and interspecific pollination studies in Populus nigra x P. nigra and P. nigra x P. deltoides, respectively. Poplar flowers were dissected into stigma, ovary and pedicel. Abscisic acid concentrations were measured at receptivity and 5 days after pollination in each part of the flower. Abscisic acid was fractionated by reverse-phase HPLC followed by an enzyme-linked immunosorbent assay (ELISA) with anti-ABA polyclonal antibody and GC-MS identification of the immunoreactive ABA fractions. Samples were fractionated and ABA was identified by the presence of a cross-reactive metabolite of ABA in the extracts. The interspecific cross (P. nigra x P. deltoides) had a high concentration of ABA in the pedicel, where abscission occurred, whereas in the intraspecific cross, ABA concentration in the pedicel was low. The results are discussed with reference to the control ABA exerts on the abscission of poplar flowers after interspecific incompatible pollinations.     

过量表达LAR3和Bbchit1以提高毛白杨对叶枯病的抗性

利用转基因技术将多种抗病基因共同转入毛白杨中以提高其抗性,从而获得毛白杨抗病新品种是目前解决杨树真菌病害的主要研究方向之一。本研究通过根癌农杆菌介导的二次遗传转化,将来源于球孢白僵菌几丁质酶基因Bbchit1转入过量表达无色花色素还原酶基因LAR3转基因毛白杨中,实时定量PCR显示Bbchit1与LAR3均能有效表达,离体抗病试验显示Bbchit1+LAR3共表达转基因毛白杨细胞粗提液对杨树叶枯病菌具有明显抑制作用,进一步将叶枯病菌接种在转基因和非转基因毛白杨叶片上培养30天,转基因植株的感病面积均低于非转基因植株且Bbchit1+LAR3共表达转基因株系抗病效果更明显。上述抗病试验结果表明:LAR3和Bbchit1在杨树中共表达可提高其对叶枯病的抗性。     

Seasonal changes in the transient expression of a 35S CaMV-GUS gene construct introduced into Scots pine buds

Seasonal changes in the transient expression of Beta-glucuronidase gene (GUS) driven by a constitutive 35S CaMV-promoter in Scots pine (Pinus sylvestris L.) buds were studied by the microprojectile DNA-delivery method. Buds were collected from 5-, 15- and 50-year-old trees. In buds from all age groups the amount of transient expression was dependent on the season; the highest values were found in March, and values were lowest both at the beginning and at the end of the growing season. Pretreatment with growth regulators increased both the amount of transient GUS expression and arginine decarboxylase (ADC) activity in buds indicating an increase in metabolic activity. These results confirm that the genetic transformation technique can be used to study seasonally dependent regulation in mature Scots pine tissues.     

Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

Compression control and its significance in the manufacture and effects on properties of poplar LVL

Summary In order to control the compression effectively, the main processing parameters for hot-pressing of poplar LVL were investigated in this study. Results from an orthogonal experiment show qualitatively that compression of poplar LVL is influenced by pressing pressure and moisture content of the veneers. High press pressure and veneer moisture content lead to high compression during hot pressing. It is shown that compression has significant effects on modulus of elasticity, modulus of rupture, specific gravity and thickness swelling of poplar LVL. Modulus of elasticity, modulus of rupture and specific gravity appear to be directly proportional to compression within the compression range of 5% to 20%. Horizontal shear strength results indicate that, due to inadequate contact, proper glue bond may not be achieved between veneers of LVL with low compression. Thickness swelling appears not sensitive to compression between the compression range of 4% to 10%.The authors wish to acknowledge the financial support from the Natural Sciences and Engineering Research Council of Canada to this study, which was carried out when the first author was a visiting scientist at Wood Science and Technology Centre, University of New Brunswick, Canada. The veneers and adhesive used in this study were provided by Temlam Inc., Ville-Marie, Quebec. Their contribution is gratefully acknowledged.     

Different sensitivity of isoprene emission, respiration and photosynthesis to high growth temperature coupled with drought stress in black poplar (Populus nigra) saplings

The effects of the interaction between high growth temperatures and water stress on gas-exchange properties of Populus nigra saplings were investigated. Water stress was expressed as a function of soil water content (SWC) or fraction of transpirable soil water (FTSW). Isoprene emission and photosynthesis (A) did not acclimate in response to elevated temperature, whereas dark (R(n)) and light (R(d)) respiration underwent thermal acclimation. R(d) was ~30% lower than R(n) irrespective of growth temperature and water stress level. Water stress induced a sharp decline, but not a complete inhibition, of both R(n) and R(d). There was no significant effect of high growth temperature on the responses of A, stomatal conductance (g(s)), isoprene emission, R(n) or R(d) to FTSW. High growth temperature resulted in a significant increase in the SWC endpoint. Photosynthesis was limited mainly by CO(2) acquisition in water-stressed plants. Impaired carbon metabolism became apparent only at the FTSW endpoint. Photosynthesis was restored in about a week following rewatering, indicating transient biochemical limitations. The kinetics of isoprene emission in response to FTSW confirmed that water stress uncouples the emission of isoprene from A, isoprene emission being unaffected by decreasing g(s). The different kinetics of A, respiration and isoprene emission in response to the interaction between high temperature and water stress led to rising R(d)/A ratio and amount of carbon lost as isoprene. Since respiration and isoprene sensitivity are much lower than A sensitivity to water stress, temperature interactions with water stress may dominate poplar acclimatory capability and maintenance of carbon homeostasis under climate change scenarios. Furthermore, predicted temperature increases in arid environments may reduce the amount of soil water that can be extracted before plant gas exchange decreases, exacerbating the effects of water stress even if soil water availability is not directly affected.     

Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic Tobacco

To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.     

Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic To-bacco

To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.     

Effects of near-lethal heat stress on bud break, heat-shock proteins and ubiquitin in dormant poplar (Populus nigra Charkowiensis x P. nigra incrassata)

We assessed the effects of near-lethal heat stress on bud break, heat-shock proteins (HSPs) and ubiquitin in hybrid poplar (Populus nigra (L.) Charkowiensis x P. nigra (L.) incrassata). Shoots, with 10-15 buds each, were collected from September to March and exposed to temperatures between 20 and 60 degrees C for 2 h. Shoots were then placed in a greenhouse at 18-22 degrees C with supplemental light and cumulative bud break was recorded over a 4-week period. Samples of bud tissues were collected during and up to 96 h after heat treatment for protein analysis. De novo synthesis of proteins was monitored by exposing excised buds to [(35)S]-methionine for 3 h before, during, or after heat treatment. Heat treatments of 40-45 degrees C resulted in both a release from endodormancy and a decrease in thermal units needed for bud break during ecodormancy. The response to near-lethal heat stress was complex and was affected by intrinsic thermal sensitivity. Heat treatments were least effective during August and became progressively more effective as endodormancy progressed. In the later stages of ecodormancy, a heat treatment of 45 degrees C either inhibited bud break or killed the buds. Although temperatures of 42.5 to 45 degrees C inhibited incorporation of [(35)S]-methionine into proteins for at least 48 h, several HSPs were synthesized in response to temperatures of 40-45 degrees C. Immunoblots indicated that one of the heat-induced proteins was immunologically related to HSP70. Increases in free and conjugated forms of ubiquitin were also observed in response to heat treatment. Production of HSPs and ubiquitin, however, was not consistently associated with the heat treatments that induced the highest percentage of bud break. The roles of heat-induced protein degradation, HSPs, and ubiquitin in overcoming dormancy by near-lethal heat stress are discussed.     

Role of phosphorus and potassium nutrition in enhancing yield,nutrient use efficiency and quality of wheat under variable aged poplar (Populus deltoides Bartr.) plantations in India

Agroforestry Systems - Light, nutrients and moisture are limiting factors altering wheat crop performance in poplar based agroforestry systems. Among these, light along with nutrients are principal...     

Key roles of leaves, stockplant age, and auxin concentration in vegetative propagation of two African mahoganies: Khaya anthotheca Welw. and Khaya ivorensis A. Chev

Leaf area, IBA concentration and age of stockplants were all found to be important factors for successful rooting for vegetative propagation using single-node cuttings of the two main African mahogany species: Khaya anthotheca and K. ivorensis. Cuttings with leaf area of 30–50 cm² had the best rooting percentage and cuttings with about 30 cm² had the most number of roots per cutting in K. anthotheca. Khaya ivorensis, cuttings with 10–30 cm² leaf area had the highest rooting percentage. Cuttings collected from 1-year-old stockplants recorded the highest rooting percentage and largest number of roots per cutting. Cuttings from 3-year-old stockplant of the same seeds sources had the lowest rooting suggesting aging negatively impacted rooting ability. The effect of auxin concentration, on rootability was examined with cuttings of K. anthotheca. An IBA concentration of 0.8% was the best exogenous auxin concentration for percentage rooting, number of roots per cutting and the length of the longest root per cutting.     

Variation in glomalin in soil profiles and its association with climatic conditions,shelterbelt characteristics,and soil properties in poplar shelterbelts of Northeast China

Glomalin-related soil protein(GRSP)sequesters large amounts of carbon and plays important roles in maintaining terrestrial soil ecosystem functions and ecological restoration;however,little is known about GRSP variation in 1-m soil profiles and its association with stand characteristics,soil properties,and climatic conditions,hindering GRSP-related degraded soil improvement and GRSP evaluation.In this study,we sampled soils from 1-m profiles from poplar(Populus spp.)shelterbelts in Northeast China.GRSP contents were 1.8–2.0 times higher in the upper 40 cm soil layers than at 40–100 cm.GRSP-related soil organic carbon(SOC)sequestration in deeper soil layers was*1.2 times higher than in surface layers.The amounts of GRSP-related nutrients were similar throughout the soil profile.A redundancy analysis showed that in both surface and deeper layers,soil properties(pH,electrical conductivity,water,SOC,and soil nutrients)explained the majority of the GRSP variation(59.5–84.2%);the second-most-important factor in GRSP regulation was climatic conditions(temperature,precipitation,and altitude),while specific shelterbelt characteristics had negligible effects(<5%).Soil depth and climate indirectly affected GRSP features via soil properties,as manifested by structural equation model analysis.Our findings demonstrate that GRSP is important for carbon storage in deep soils,regardless of shelterbelt characteristics.Future glomalin assessments should consider these vertical patterns and possible regulating mechanisms that are related to soil properties and climatic changes.     

Performance and variability patterns in wood properties and growth traits in the parents, F1 and F2 generation hybrid clones of Populus deltoides

The performance and variability patterns in the wood element’s dimensions, specific gravity and growth parameters namely ramet height and GBH were evaluated in 16 clones of parents, F1 and F2 hybrids of Populus deltoides Bartr. ex Marsh. Ramet radial variations were nonsignificant, while inter-clonal variations due to interaction of clone/replication were significant for all the wood traits except vessel element length. Inter-clonal variations were significant only for fiber length and fiber wall thickness....     

Cryphonectria parasitica in Portugal: diversity of vegetative compatibility types,mating types,and occurrence of hypovirulence

Cryphonectria parasitica, the causal agent of chestnut blight, has been responsible for the decline in chestnut in Portugal for the last two decades. In order to improve understanding of C. parasitica diversity, a total of 617 isolates from all affected chestnut‐growing areas in continental Portugal, Madeira and the Azores archipelagos were studied. Nine vegetative compatibility (vc) types were identified among the isolates. EU‐11 was the most widespread vc type comprising 80.2% of the isolates, followed by EU‐12 (7.1%) and EU‐66 (6.6%). Two of the Portuguese vc types could not be assigned to a known European vc type. The diversity of vc types was low in the Portuguese populations of C. parasitica, but comparable with other areas where C. parasitica was introduced recently. The frequent occurrence of perithecia and both mating types of C. parasitica indicates that sexual reproduction of the chestnut blight fungus is common in Portugal. One C. parasitica isolate from Trás‐os‐Montes showed a white culture morphology and contained dsRNA, indicating the presence of hypovirulence in this area.