Inheritance and molecular variations of PCR-SSCP fragments in pedunculate oak (Quercus robur L.)

Single-strand conformaiton polymorphism (SSCP) profiles of six PCR-amplified fragments (250–800 bp) were analyzed in three full-sib families of pedunculate oak (Quercus robur L.) and their parents. Among the six fragments, four were polymorphic and one exhibited complex patterns that were not changed by varying the SSCP conditions. The number of bands for the analyzed fragments varied between two and four among individuals regardless of fragment size. As shown by segregation data, the variation in the number of bands between trees could only be attributed to the allelic composition (homozygotes vs heterozygotes): a genotype that exhibited two bands was presumptively homozygous, wheras a genotype exhibiting three or four bands was heterozygous. Mendelian proportions were observed in all crosses for each polymorphic fragment. In one cross, we could clearly identify a null allele due to a possible mutation at a primer site. Single-base mutations and short insertion-deletions were shown to be the molecular causes of the SSCP polymorphism observed between different alleles. The use of SSCP as a technique to identify co-dominant markers of PCR fragments (up to 800 bp) is recommended for gene diversity studies or for gene mapping.     

Structure and identification of root bark of Quercus robur L.

The root bark structure of Quercus robur L. was analysed at different stages of root development and compared to the structure of stem bark. Root bark thickness varied considerably between different roots. Sclereid quantity decreased with increasing distance from the stem, which means it increased with age. Visible growth increments diminished with increasing distance from the stem. In lateral roots crystal quantity decreased with increasing distance from the stem. In lateral roots secondary phloem fibre length, sieve tube member length, and sieve tube diameter showed no regular trend. There were only a few basic structural differences between root and stem bark. The zone of cell differentiation (cell expansion, lignification) was wider in root bark; sieve tube collapse was delayed. In lateral root bark fewer sclereids were formed. The first-formed periderm often originated from deeper cell layers. Thus, primary elements were lacking after periderm formation. In root bark the phellem cell walls were of equal thickness. Thus, phellem lacked visible growth increments. Root bark phellem cells were slightly larger. The root phelloderm was more distinct. The secondary phloem fibres were slightly shorter than those in stem bark. Sieve tube members of stem and root bark were of similar length and diameter. The qualitative bark anatomical characters of oak root bark are suitable for root identifications. Due to minor structural differences between root and stem bark the characters must be used with care.     

Transport of organic sulfur and nitrogen in the roots of young mycorrhizal pedunculate oak trees (Quercus robur L.)

Uptake and xylem loading of organic sulfur and nitrogen were analyzed in detached mycorrhizal (Laccaria laccata L.) roots of pedunculate oak (Quercus robur L.) seedlings using radiolabeled reduced glutathione (GSH) and glutamine (Gln) for transport analyses. The experiments showed for the first time that GSH is taken up by plant roots from the nutrient solution and is partially allocated to the shoot. Apparently, GSH produced during mineralization processes in the soil can be used by plant roots as a sulfur source. GSH uptake into the roots showed biphasic kinetics within the concentration range studied (0–500 M) with maximum transport velocities (v _max) and substrate affinities (K _m) that were similar to the kinetics of Gln uptake. GSH uptake kinetics were also in the same range as previously reported for sulfate uptake by mycorrhizal roots of pedunculate oak. It may therefore be assumed that GSH and sulfate uptake can be of comparable significance for sulfur nutrition, provided both sulfur sources are available at similar concentrations at the sites of uptake. Xylem loading of GSH and Gln showed monophasic transport kinetics with v _max significantly lower than observed for the two respective uptake systems and, as indicated by the K _m-values, a substrate affinity between the high and the low affinity uptake systems. The possible nature of the transport systems for GSH and Gln is discussed.     

Radial distributions of Cd in stems of oak trees (Quercus robur L.) re-analyzed after 10 years

Radial distribution patterns of Cd were determined in stems of the same oak trees (Quercus robur L.) in 1983 and again in 1994. On both sampling dates the same distribution patterns were generally observed. Highest concentrations of Cd were found at the sapwood-heart-wood transition. A sharp drop over this boundary towards inner parts of the stems occurred in all trees. In the decade between the two investigations the sapwood-heartwood boundaries had shifted outwards by 9–11 annual rings. The Cd peaks at the boundaries were shifted by approximately the same interval. The results suggest that the described Cd peaks are mobile in a radial direction. The present location of such peaks cannot be used to infer the pollution history of the tree's environment. The Cd accumulation at the sapwood-heartwood boundary is probably affected by physiological processes in the wood. Thus it is concluded that radial distributions of Cd in stems of oak trees are no reliable source of information for retrospective monitoring of past time pollution levels.     

Influence of wind loading on root system development and architecture in oak (Quercus robur L.) seedlings.

The effect of wind loading on seedlings of English oak (Quercus robur L.) was investigated. Instead of using a traditional wind tunnel, an innovative ventilation system was designed. This device was set up in the field and composed of a rotating arm supporting an electrical fan, which emitted an air current similar to that of wind loading. Oaks were sown from seed in a circle around the device. A block of control plants was situated nearby, and was not subjected to artificial wind loading. After 7 months, 16 plants from each treatment were excavated, and root architecture and morphological characteristics measured using a 3D digitiser. The resulting geometrical and topological data were then analysed using AMAPmod software. Results showed that total lateral root number and length in wind stressed plants were over two times greater than that in control trees. However, total lateral root volume did not differ significantly between treatments. In comparing lateral root characters between the two populations, it was found that mean root length, diameter and volume were similar between the two treatments. In trees subjected to wind loading, an accentuated asymmetry of root distribution and mean root length was found between the windward and leeward sides of the root system, with windward roots being significantly more numerous and longer than leeward roots. However, no differences were found when the two sectors perpendicular to the wind direction were compared. Mean tap root length was significantly higher in control samples compared to wind stressed plants, whilst mean diameter was greater in the latter. Wind loading appears to result in increased growth of lateral roots at the expense of the tap root. Development of the lateral root system may therefore ensure better anchorage of young trees subjected to wind loading under certain conditions.     

Global 5-methylcytosine alterations in DNA during ageing of Quercus robur seeds

Background and Aims Epigenetic regulation plays an important role in the management of plant growth, development and response to stress factors, and several reports have indicated that DNA methylation plays a critical role in seed development and viability. This study examines changes in 5-methylcytosine (m⁵C) levels in the DNA of seeds during ageing, a process that has important implications for plant conservation and agriculture.Methods Changes in the global level of m⁵C were measured in mature seeds of oak, Quercus robur. The extent of DNA methylation was measured using a protocol based on two-dimensional thin-layer chromatography. Viability of seeds was determined by germination and seedling emergence tests.Key Results An ageing-related decrease in total m⁵C during storage of recalcitrant seeds was highly and significantly correlated with a decrease in seed viability, as reflected by a reduction in germination (r = 0·8880) and seedling emergence (r = 0·8269).Conclusions The decrease in viability during ageing of Q. robur seeds is highly correlated with a global decline in the amount of m⁵C in genomic DNA, and it is possible that this may represent a typical response to ageing and senescence in recalcitrant seeds. Potential mechanisms that drive changes in genomic DNA methylation during ageing are discussed, together with their implications for seed viability.     

Quantitative trait loci controlling water use efficiency and related traits in Quercus robur L.

Genetic variation for intrinsic water use efficiency (W _i) and related traits was estimated in a full-sib family of Quercus robur L. over 3 years. The genetic linkage map available for this F1 family was used to locate quantitative trait loci (QTL) for W _i, as estimated by leaf carbon stable isotope composition (δ ¹³C) or the ratio of net CO₂ assimilation rate (A) to stomatal conductance to water vapour (g _w) and related leaf traits. Gas exchange measurements were used to standardize estimates of A and g _w and to model the sensitivity of g _w to leaf-to-air vapour pressure deficit (sg^VPD). δ ¹³C varied by more than 3‰ among the siblings, which is equivalent to 40% variation of W _i. Most of the studied traits exhibited high clonal mean repeatabilities (>50%; proportion of clonal mean variability in global variance). Repeatabilities for δ ¹³C, leaf mass per area (LMA) and leaf nitrogen content were higher than 70%. For δ ¹³C, ten QTLs were detected, one of which was detected repeatedly for all 3 years and consistently explained more than 20% of measured variance. Four genomic regions were found in which co-localizing traits linked variation in W _i to variations in leaf chlorophyll and nitrogen content, LMA and sg^VPD. A positive correlation using clonal means between δ ¹³C and A/g _w, as well as a co-localisation of QTL detected for both traits, can be seen as validation of the theoretical model linking the genetic architecture of these two traits.     

Heterologous expression of functionally active enterolysin A, class III bacteriocin from Enterococcus faecalis, in Escherichia coli

The heterologous expression of enterolysin A (EnlA), heat-labile class III bacteriocin from Enterococcus faecalis II/1 with anti-listerial activity, was studied in Escherichia coli. The PCR amplified products of enterolysin A structural gene, N-terminal part of EnlA with endopeptidase-like activity and C-terminal part of EnlA similar to a lysis gene of bacteriophage, were cloned in prelinearized pQE-30UA expression vector. The expression of EnlA structural gene led to the synthesis and secretion of functional-active His-tagged enterolysin A protein, which was purified to homogeneity using His-Select™ Cartridge and was shown to be fully active against the indicator strain. The expression of N-terminal or C-terminal part of EnlA and deletion of last 58 amino acids from C-terminal domain of EnlA led to the synthesis of biologically non-active proteins.     

Influence of root temperature on growth of Pinus sylvestris,Fagus sylvatica,Tilia cordata and Quercus robur

One-year-old tree seedlings were incubated in a greenhouse from April to July, under natural daylight conditions, with their root systems at constant temperatures of 5, 10, 15, 20, 25, 30 and 35 °C and with the above ground parts kept at a constant air temperature of 18–20 °C. The course of height growth, total mass increment, root, shoot and leaf weight as well as leaf areas were measured. The results indicate that clear differences exist in the optimal root zone temperatures for various growth parameters in different tree species. Pinus sylvestris had a maximal height increment at about 5–10 °C and maximal total mass increment at 15 °C root temperature. In contrast, the optimum for Quercus robur was at 25 °C. Tilia cordata and Fagus sylvatica had their optima for most growth parameters at 20 °C. The root temperature apparently indirectly influenced photosynthesis (dry weight accumulation) and respiration loss. From the observed symptoms and indications in the literature it seems probable that a change in hormone levels is involved as the main factor in the described effects. Variation of root temperature had only an insignificant effect on bud burst and the time at which the shoots sprouted. Apparently species of northern origin seem to have lower root temperature optima than those of more southern origin. This is to be verified by investigation of other tree species.     

Interactive effects of mycorrhization and elevated carbon dioxide on growth of young pedunculate oak (Quercus robur L.) trees

Pedunculate oak (Quercus robur L.) was germinated and grown at ambient CO₂ level and 650 ppmv CO₂ in the presence and absence of the ectomycorrhizal fungus Laccaria laccata for a total of 6 month under nutrient non-limiting conditions. Mycorrhization and elevated atmospheric CO₂ each supported the growth of the trees. Stem height, stem diameter, and dry matter accumulation of pedunculate oak were increased by mycorrhization. Elevated atmospheric CO₂ enhanced stem height, stem diameter, fresh weight and dry weight, as well as lateral root formation of the trees. In combination, mycorrhization and elevated atmospheric CO₂ had a more than additive, positive effect on tree height and biomass accumulation, and further improved lateral root formation of the trees. From these findings it is suggested that the efficiency of the roots in supporting the growth of the shoot is increased in mycorrhized oak trees at elevated atmospheric CO₂.Abbreviations DW dry weight - FW fresh weight - RWC relative water content     

The alpha-tocopherol content of leaves of pedunculate oak (Quercus robur L.)--variation over the growing season and along the vertical light gradient in the canopy

This study was performed in order to investigate whether the actual requirement for defence against photo-oxidative stress is reflected by the alpha-tocopherol (alpha-Toco) content in leaves of pedunculate oak (Quercus robur L.). Antioxidants and pigments were quantified in leaves that were collected on six days between May and September 2000 in a mixed pine/oak forest at canopy positions differing in light environment. Pools of hydrophilic antioxidants and photo-protective xanthophyll cycle pigments (V + A + Z) reflected the anti-oxidative demand, as these pools increased with the average light intensity to which the leaves were acclimated. The photo-protective demand was not the determinant of the alpha-Toco content of oak leaves, as (1) foliage of a young oak, exposed to low light levels in the understorey, contained higher amounts of this lipophilic antioxidant than leaves sampled from semimature oaks at canopy positions with a similar light environment, and (2) a strong increase in the alpha-Toco content over the growing season was detected at each investigated crown position, whereas the V + A + Z pool did not show a concomitant accumulation during leaf ageing. The rate of alpha-Toco accumulation differed distinctly between samples taken at different canopy positions.     

Substrate specificity and antifungal activity of recombinant tobacco class I chitinases

Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.     

Flooding effects on starch partitioning during early growth of two oak species

Pedunculate (Quercus robur L.) and sessile (Q. petraea [Matt.] Liebl.) oaks are the most common oak species in Western Europe. They are known to display different ecological requirements, particularly relative to root hypoxia induced by flooding: In a glasshouse study of seedlings, we quantified the effects of flooding on starch mobilization from cotyledons and starch partitioning. Growth and distribution of lateral roots were also measured. The above-ground growth of Q. robur was less affected by flooding than that of Q. petraea which failed to develop a second flush. Root growth was also severely inhibited, particularly in Q. petraea. In Q. robur, lateral root initiation as well as elongation was restricted to the soil surface layer. Flooding markedly reduced total growth and concentrations of in all components except stems. Starch mobilization from cotyledons was delayed by flooding, especially in Q. robur seedlings. Under flooding, the decrease of cotyledons dry mass and starch content in Q. robur was lower than in Q. petraea, whereas Q. robur displayed larger growth than Q. petraea. The features of carbohydrate management may be crucial in the observed differences in flooding tolerance of these species.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Ectomycorrhiza communities of red oak (<Emphasis Type="BoldItalic">Quercus rubra</Emphasis> L.) of different age in the Lusatian lignite mining district,East Germany

Ectomycorrhizal (ECM) communities were assessed on a 720 m² plot along a chronosequence of red oak (Quercus rubra) stands on a forest reclamation site with disturbed soil in the lignite mining area of Lower Lusatia (Brandenburg, Germany). Adjacent to the mining area, a red oak reference stand with undisturbed soil was investigated reflecting mycorrhiza diversity of the intact landscape. Aboveground, sporocarp surveys were carried out during the fruiting season in a 2-week interval in the years 2002 and 2003. Belowground, ECM morphotypes were identified by comparing sequences of the internal transcribed spacer regions from nuclear rDNA with sequences from the GenBank database. Fifteen ECM fungal species were identified as sporocarps and 61 belowground as determined by morphological/anatomical and molecular analysis of their ectomycorrhizas. The number of ECM morphotypes increased with stand age along the chronosequence. However, the number of morphotypes was lower in stands with disturbed soil than with undisturbed soil. All stands showed site-specific ECM communities with low similarity between the chronosequence stands. The dominant ECM species in nearly all stands was Cenococcum geophilum, which reached an abundance approaching 80% in the 21-year-old chronosequence stand. Colonization rate of red oak was high (>95%) at all stands besides the youngest chronosequence stand where colonization rate was only 15%. This supports our idea that artificial inoculation with site-adapted mycorrhizal fungi would enhance colonization rate of red oak and thus plant growth and survival in the first years after outplanting.     

The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P < 0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts.