Leaf structural modifications in Populus × euramericana subjected to Zn excess

In previous experiments elevated but sub-symptomatic applications of Zn (0.1 mM and 1 mM) caused impairments in growth parameters and photosynthetic performance of Populus × euramericana (Dode) Guinier clone I-214. The aim of this work was to evaluate leaf morphological and anatomical traits in this clone in response to the same Zn concentrations. The results showed that Zn treatments induced variations in leaf dry mass, area, mesophyll thickness, intercellular spaces, stomatal density and size. Stronger modifications, especially concerning stomata characteristics induced by 1 mM Zn, were consistent with physiological impairments while those induced by 0.1 mM Zn suggested a compensatory strategy for maintaining functional integrity.     

Ozone-induced microscopical changes and quantitative carbohydrate contents of hybrid poplar (Populus × euramericana)

Summary Cuttings of hybrid poplar (Populus × euramericana var. Dorskamp) were exposed to ozone (80 g/m³ from 2100 hours to 0700 hours, 180 g/m³ from 0700 hours to 2100 hours) for 3 months. Ozone reduced the starch content in leaves and stem bark, whereas starch granules accumulated in bundle sheath cells along small leaf veins. At the same time, sucrose and inositol content increased in the leaves. Mesophyll cells in the vicinity of the stomata were injured first, and droplet-like material appeared on their walls. In the sieve plates of fumigated trees, the pores showed a higher degree of narrowing than those of the control treatment. Cell collapse in the leaves was accompanied by water loss and an increase in air space. In the stems, the ozone treatment led to a reduced radial width, particularly in the xylem tissue. These results are discussed in relation to reduced or inhibited phloem loading and ozone-induced drought stress. The plants injured by ozone showed quite distinct patterns of metabolite responses as well as enzyme activities (PEP- and RubP-carboxylase) in the leaves from the top to the bottom. There were also remarkable differences in the reaction of sucrose and inositol between leaves and stem bark. Future research should therefore increasingly follow a whole-plant approach for a better understanding of complex plant reactions.     

Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus × euramericana 'Guariento')

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.     

The effect of methyl salicylate on the induction of direct and indirect plant defense mechanisms in poplar (Populus × euramericana ‘Nanlin 895’)

The objective of this study was to investigate the effect of different concentrations of methyl salicylate (MeSA) on direct defense and indirect defense in poplar cuttings (Populus × euramericana ‘Nanlin 895’). Four defense-related enzyme activities, such as superoxide dismutase (SOD, EC1.15.1.1), oxydoreductases peroxidase (POD, EC1.11.1.7), catalase (CAT, EC1.11.1.6), and polyphenol oxidase (PPO, EC 1.14.18.1), were measured. The results showed that the SOD activities were induced by 1.0 and 10.0 mM MeSA and the POD activities were induced by MeSA. The CAT activity was induced at low concentrations of MeSA but was inhibited by high concentrations, and the PPO activity was affected by 0.1, 1.0, and 10.0 mM MeSA. Furthermore, the volatiles were detected using gas chromatography–mass spectrometry and gas chromatography. Twenty-one volatile compounds were tentatively identified in the emissions from plant leaves. Of these volatile compounds, (Z)-3-hexen-1-ol and cis-3-hexenyl acetate emissions were the highest. (Z)-3-Nonen-1-ol, (E)-2-hexen-1-ol, 1-octanol, (E,Z)-3,6-nonadien-1-ol, β-ionone, and hexadecanamide were six compounds that were only produced after treatment by 10.0 mM MeSA. These results showed that direct and indirect defense mechanisms in poplars were induced by MeSA.     

Hypoxia Affects Nitrogen Uptake and Distribution in Young Poplar (Populus × canescens) Trees

The present study with young poplar trees aimed at characterizing the effect of O₂ shortage in the soil on net uptake of NO₃ ^- and NH₄ ⁺ and the spatial distribution of the N taken up. Moreover, we assessed biomass increment as well as N status of the trees affected by O₂ deficiency. For this purpose, an experiment was conducted in which hydroponically grown young poplar trees were exposed to hypoxic and normoxic (control) conditions for 14 days. ¹⁵N-labelled NO₃ ^- and NH₄ ⁺ were used to elucidate N uptake and distribution of currently absorbed N and N allocation rates in the plants. Whereas shoot biomass was not affected by soil O₂ deficiency, it significantly reduced root biomass and, consequently, the root-to-shoot ratio. Uptake of NO₃ ^- but not of NH₄ ⁺ by the roots of the trees was severely impaired by hypoxia. As a consequence of reduced N uptake, the N content of all poplar tissues was significantly diminished. Under normoxic control conditions, the spatial distribution of currently absorbed N and N allocation rates differed depending on the N source. Whereas NO₃ ^- derived N was mainly transported to the younger parts of the shoot, particularly to the developing and young mature leaves, N derived from NH₄ ⁺ was preferentially allocated to older parts of the shoot, mainly to wood and bark. Soil O₂ deficiency enhanced this differential allocation pattern. From these results we assume that NO₃ ^- was assimilated in developing tissues and preferentially used to maintain growth and ensure plant survival under hypoxia, whereas NH₄ ⁺ based N was used for biosynthesis of storage proteins in bark and wood of the trees. Still, further studies are needed to understand the mechanistic basis as well as the eco-physiological advantages of such differential allocation patterns.     

Haploid plants from anther cultures of poplar (Populus × beijingensis)

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial woody plants using conventional breeding techniques is currently a challenge due to a long juvenile period, high heterozygosity, and substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In the present study, we report the regeneration of haploid lines of poplar (Populus × beijingensis) via anther culture. Anthers at the uninucleate stage were induced to produce callus using three basic media. Two auxins (naphthalene acetic acid [NAA] and 2,4-dichloro-phenoxyacetic acid [2,4-D]), and two cytokinin (kinetin [KT] and 6-benzyladenine [BA]) were tested to explore the influence of plant growth regulators on callus response. H medium (Bourgin and Nitsch 1967) supplemented with 1.0 mg/L NAA and 1.0 mg/L KT induced the highest rate of callus formation. When callus obtained from anthers were subcultured in MS medium containing 1.0 mg/L BA and 0.2 mg/L NAA, followed by transfer to half-strength MS medium supplemented with indole-3-butyric acid (0.2–0.5 mg/L), the formation of regenerated plantlets increased dramatically. Inclusion of gibberellic acid (0.02–0.2 mg/L) in addition to a combination of BA (0.6 mg/L)-NAA (0.2 mg/L) in the culture medium resulted in enhanced frequency of shoot development, as well as greater internode elongation. Ploidy analysis of 580 regenerated plants, using both flow cytometry and chromosome counting, revealed 10.3 % haploid and 1.0 % triploid plantlets. The remaining plantlets were all diploid.     

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.     

Agrobacterium strain specificity and shooty tumour formation in eucalypt (Eucalyptus grandis ×E. urophylla)

Summary To develop a successful protocol forAgrobacterium-mediated transformation in plants it is essential to determine the most efficient bacterial strain/plant genotype interaction. In the present work, we evaluated the susceptibility ofEucalyptus grandis ×E. urophylla to fiveAgrobacterium rhizogenes and twelveA. tumefaciens wildtype strains. The results showed different degrees of virulence, according to the strain tested, indicating that transformation of this eucalypt hybrid by Agrobacterium-derived vectors is possible. All developed tumours showed an autonomous growth when transferred to a hormone-free medium. Some of these tumours formed shoots spontaneously, with a normal phenotype. Polymerase Chain Reaction (PCR) and Southern blot analyses were performed to confirm the absence of the oncogenic T-DNA in plants derived from these shooty tumours.     

Is egg carrying attractive? Mate choice in the golden egg bug (Coreidae,Heteroptera)

In several species of fish, females select males that are already guarding eggs in their nests. It is a matter of debate as to whether a female selects a good nest site for her offspring (natural selection) or a male for his attractiveness (sexual selection). The golden egg bug, Phyllomorpha laciniata Vill, resembles fish in the sense that mating males carry more eggs than single males, but in the bugs, female mate choice is decoupled from egg site choice. The sexual selection hypothesis predicts that if females select males using male egg load as a cue for male quality, they should not mate with a male when eggs are removed, regardless of his mating attempts. When individual females were enclosed with an egg-loaded male and an unloaded male, they mated equally often with both males, although the loaded males courted more. In addition, when only successful males were used, females mated equally often with the loaded male and the unloaded male irrespective of sex ratio. Male choice rather than female choice affected mating frequency when sex ratio was equal. Therefore, females do not select the male by the eggs he carries, but successful males may receive many eggs due to egg dumping by alien females while they mate or as a consequence of mate guarding.     

Which egg features predict egg rejection responses in American robins? Replicating Rothstein's (1982) study

Rothstein (Behavioral Ecology and Sociobiology, 11, 1982, 229) was one of the first comprehensive studies to examine how different egg features influence egg rejection behaviors of avian brood parasite–hosts. The methods and conclusions of Rothstein (1982) laid the foundation for subsequent experimental brood parasitism studies over the past thirty years, but its results have never been evaluated with replication. Here, we partially replicated Rothstein's (1982) experiments using parallel artificial model egg treatments to simulate cowbird (Molothrus ater) parasitism in American robin (Turdus migratorius) nests. We compared our data with those of Rothstein (1982) and confirmed most of its original findings: (1) robins reject model eggs that differ from the appearance of a natural robin egg toward that of a natural cowbird egg in background color, size, and maculation; (2) rejection responses were best predicted by model egg background color; and (3) model eggs differing by two or more features from natural robin eggs were more likely to be rejected than model eggs differing by one feature alone. In contrast with Rothstein's (1982) conclusion that American robin egg recognition is not specifically tuned toward rejection of brown‐headed cowbird eggs, we argue that our results and those of other recent studies of robin egg rejection suggest a discrimination bias toward rejection of cowbird eggs. Future work on egg recognition will benefit from utilizing a range of model eggs varying continuously in background color, maculation patterning, and size in combination with avian visual modeling, rather than using model eggs which vary only discretely.     

Barley Chromosome Identification Using Genomic in Situ Hybridization in the Genome of Backcrossed Progeny of Barley–Wheat Amphiploids [Hordeum geniculatum all. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70)

Genomic in situ hybridization (GISH) has been used to study characteristics of the formation of alloplasmic lines detected among self-pollinated backcrossed progeny (BC₁F₅–BC₁F₈) of barley–wheat amphiploids [Hordeum geniculatum All. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70). The chromosome material of the wild barley H. geniculatum has been shown to contribute to these lines. For example, fifth-generation plants (BC₁F₅) had genotypes (2n= 42w + 2g), (2n = 42w + 1g + 1tg), and (2n = 41w + 1g), where w is common wheat chromosomes, g is barley (H. geniculatum) chromosomes, and tg is the telocentric chromosome of wild barley. Beginning from theBC₁F₆ generation, alloplasmic telocentric addition lines (2n= 42 + 2tg) and (2n = 42 + 1tg) appear. This lines has been found cytogenetically unstable. The progeny of each of these cytological types include not only the (2n= 42 + 2tg) and (2n = 42 + 1tg) addition plants, but also plants with the monosomic (2n = 41 + 1tg) and the disomic (2n = 40 + 2tg) substitutions, as well as the (2n = 41 + 2tg) plants, which lack one wheat chromosome and have two telocentric barley chromosomes. It has been demonstrated that the selection for well-filled grains favors the segregation of telocentric addition lines (2n = 42 + 2tg) and (2n = 42 + 1tg).     

Expression of Fertility during Morphogenesis in Self-Pollinated Backcrossed Progenies of Barley—Wheat Amphiploids [Hordeum geniculatum All. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70)

The fertility characteristics expressed during morphogenesis in first-generation self-pollinated backcrossed progenies (BC₁) obtained from amphiploid barley–wheat hybrids [Hordeum geniculatum All. (2n= 28) ×Triticum aestivum L. (2n= 42)] (2n = 70) backcrossed with common wheat were studied. It was found that, in the case of self-pollination of BC₁ plants, karyotype stabilization leads to the formation of alloplasmic euploid (2n = 42), telocentric substitution (2n = 40 + 2t), and telocentric addition (2n = 42 + t), (2n = 42 + 2t) plant forms, which may serve as the sources of the respective alloplasmic lines of common wheat. That the expression of fertility characters in BC₁F₈plants was shown to depend on growth conditions. The main mechanism of hybrid incompatibility of BC₁F₁–BC₁F₈plants was expressed as grass-clump dwarfism.     

Influence of over-expression of the FLOWERING PROMOTING FACTOR 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.)

Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.     

Synaptonemal complex formation in hybrids of Lolium temulentum × Lolium perenne (L.)

Chromosome pairing and synaptonemal complex formation at zygotene and pachytene are described from serial section reconstructions of pollen mother cell nuclei in a triploid hybrid containing two haploid sets of Lolium perenne chromosomes, one of L. temulentum and two acces-sory B chromosomes. At pachytene the homologous L. perenne chromosomes form complete and continuous synaptonemal complexes while the L. temulentum chromosomes show extensive nonhomologous pairing both within and between themselves. At zygotene however, homoeologous pairing in the form of a trivalent and very little non-homologous pairing is observed. Evidently, there exists a mechanism that eliminates homoeologous association during zygotene to ensure strict bivalent formation between homologous chromosomes at pachytene. In Lolium this mechanism is under the influence of the B chromosomes and bears close similarity with that in allohexaploid wheat controlled by the Ph locus.     

Synaptonemal complex formation in hybrids of Lolium temulentum × Lolium perenne (L.)

The chromosomes of Lolium temulentum are longer and contain on average 50% more nuclear DNA than the chromosomes of L. perenne. In the hybrid, despite the difference in length and DNA content, pairing between the homoeologous chromosomes at pachytene is effective and the chiasma frequency at first metaphase in pollen mother cells is high, about 1.6 per bivalent, comparable to that in the L. perenne parent. Electron microscopic observations from reconstructed nuclei at pachytene show that synaptonemal complex (SC) formation in 40% of bivalents is perfect, complete and continuous from telomere to telomere. In others, SCs extend from telomere to telomere but incorporate lateral component loops in interstitial chromosome segments. Even in these bivalents, however, pairing is effective in the sense of chiasma formation. The capacity to form perfect SCs is achieved by an adjustment of chromosome length differences both before and during synapsis. Perfect pairing and SC formation is commoner within the larger bivalents of the complement. At zygotene, in contrast to pachytene, pairing is not confined to homoeologous chromosomes. On the contrary there is illegitimate pairing between non-homologous chromsomes resulting in multivalent formation. There must, therefore, be a mechanism operative between zygotene and pachytene that corrects and modifies associations in such a way as to restrict the pairing to bivalents comprised of strictly homoeologous chromosomes. Such a correction bears comparison with that known to apply in allopolyploids. In the hybrid and in the L. perenne parent also, certain specific nucleolar organisers are inactivated at meiosis.