Application of the RLGS Method to Large-Size Genomes Using a Restriction Trapper

We developed a method for producing restriction landmark genomicscanning (RLGS) profiles of large size genomes, such as thoseof higher plants or amphibians using a restriction trapper.Use of the conventional RLGS method is limited to genomes smallerthan 3 x 10⁹ bp, because the larger genomic DNAs, especiallythose of more than 1 x 10¹⁰ bp, produce high background dueto incorporation of radioactivity at non-specifically damagedsites. Our new method reduces the background levels by reducinggenome complexity to 1/200–1/300 using a purificationstep to enrich DNA fragments carrying specific restriction landmarksat their ends using a restriction trapper. This step makes itpossible to obtain RLGS patterns of larger genomes. Our paperdescribes the practical application for the RLGS method usinga restriction trapper with the pine tree genome (3 x 10¹⁰ bp/haploidgenome; Pinus koraiensis Sieb. et Zucc.) as an example.     

Secondary succession and effects of clear-logging on diversity in the subtropical forests on Okinawa Island,southern Japan

In order to clarify the recovery process of the subtropical forest on Okinawa Island, southern Japan, biomass accumulation and the successional trend of species diversity with time were investigated by comparing plots of old-growth and clear logged secondary forests. Self-thinning was an important factor in the development of young secondary forests, and the small variance in tree size within a stand was related to the stand not being fully stratified after clear-cutting. A large variance of size structure in old secondary and old-growth forests implies re-initiation of the understorey. Additionally, the trajectory of stand development indicated that the subtropical forest quickly recovered aboveground biomass, which reached its upper limit at about 50 years after disturbance. However, there was a large distinction in species diversity between the secondary forests and old-growth forests. The diversity of forest floor plants did not recover well after being clear-cut. This indicates that management of the subtropical forest should not only take timber-oriented tree species into account, but also the biodiversity in ground flora. The secondary forests were characterized by Castanopsis sieboldii and Schima wallichii, and the monopolization of C. sieboldii through secondary succession had a negative influence on species diversity. Distylium racemosum dominated at the late development stage and was considered a long-lived competing species that reduced the dominance of C. sieboldii and facilitated the co-occurrence of understorey species. Light-demanding pioneer tree species such as S. wallichii that regenerated after logging should be replaced by competitive effects of climax species, and thus relayed floristic change might increase species diversity along secondary succession.Nomenclature: Hatushima and Amano (1994).     

Reduction of geitonogamy: Flower abscission for departure of pollinators

Overproduction of flowers increases the attractiveness of a plant to pollinators, but results in increased geitonogamy. In general, flower abscission has been considered to be an event subsequent to the overproduction of flowers. We observed pollinator behavior in Tilia, a self-incompatible, insect-pollinated tree. We found that pollinators sequentially visited inflorescences within a tree; however, when they met with flower abortion by abscission, they were apt to move long distances and leave the tree. We propose that plants may utilize flower abscission as a method for regulating the movements of pollinators by disturbing them in order to prevent geitonogamy.     

Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere

An enzymatic method for measuring the O(2) concentrations of aqueous solutions was developed by involving 4-chlorocatechol and catechol 2,3-dioxygenase from Pseudomonas putida. With this system, the amount of O(2) in a sample solution can be measured as the amount of 5-chloro-2-hydroxymuconate semialdehyde formed through the enzyme reaction. The product was stable and its anion exhibited strong absorption around 380 nm (molar absorption coefficient of 4.3 x 10(4) M(-1) cm(-1), pK value of 5.4). A sensitive HPLC method involving a BioAssist Q column was developed to individually quantify the products derived from 4-chlorocatechol and catechol. When the O(2) concentration in a sample solution sealed in a vial was lowered from the air-saturation level by means of the amount enzymatically reacted with a known amount of catechol, the concentration of remaining O(2) could be successfully measured by the HPLC method. We developed devices through which reagents could be added to solutions sealed in cuvettes or the vessel of an oxygen electrode system under a flow of argon. By applying these devices, the submicromolar O(2) concentration of an anoxic solution and the back diffusion of O(2) from the atmosphere could be directly determined for the first time. The K(m) values of the dioxygenase and an ascorbate oxidase for oxygen were also determined to be 7.2 (at pH 7.5) and 114 microM (at pH 6.5), respectively, at 25 degrees C.     

Drebrin E is involved in the regulation of axonal growth through actin–myosin interactions

Drebrin is a well-known side-binding protein of F-actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F-actins and microtubules overlapped. Over-expression of drebrin E caused drebrin E and F-actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F-actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over-expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin–myosin interactions in the transitional zone of axonal growth cones.     

Crown hollowing as a consequence of early shedding of leaves and shoots

Leafing pattern has long been considered as an important element characterizing the growth strategy of tree species; however, the consequences of leafing pattern for tree-crown formation have not been fully understood. To address this issue, the dynamic events (growth, birth, and death) of current-year shoots and leaves were investigated together with their location in saplings of a pioneer tree, Alnus sieboldiana. The leafing pattern was characterized by successive emergence and shedding of short-lived leaves. The combination of successive leafing and within-crown variation in leaf production brought about characteristic outcomes in crown morphology. In the outer crown, because of continuous leaf production, the shoots achieved great extension and enormous daughter shoot production, resulting in rapid expansion of the crown. In contrast, in the inner crown, due to early termination of leaf production, the shoots completely lost their leaves early in the growing season and consequently themselves died and were shed within the season. Such quick shedding of shoots caused “crown hollowing”, i.e., the interior crown consisted of primary branches with little secondary development or foliage. These dynamic features are an effective adaptive strategy in early succession but also may be a disadvantage to maintaining foliage for longer period. Crown maintenance associated with the longevity of structural components is thought to play an important role in survival strategy of tree species.     

Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity

Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.     

Long-term growth dynamics of natural forests in Hokkaido,northern Japan

Abstract. The long-term growth dynamics of natural forest stands on the island of Hokkaido were described on the basis of an analysis of data from 38 permanent plots spanning 15–22 yr. Stand structure was characterized by basal area, stem density and tree size variability. To detect trends in stand structure, regression models for recruitment rate (per ha per yr), mortality rate and the rate of change in stem density and tree size variability were developed by a stepwise method using initial basal area, stem density, tree size variability, species composition summarized by LNMDS ordination, altitude, annual mean temperature, annual precipitation, type of understorey vegetation, topography and slope aspect as candidates for predictor variables. The same analyses were conducted for basal area increment (net growth) and its components: survivor growth = basal area gain by growth of surviving individuals and mortality = basal area loss by death of individuals. Stem density remained generally unchanged; recruitment was relatively low even in very sparse stands. Stand basal area generally increased as survivor growth was approximately double the mortality. Recruitment rate was strongly affected by the presence of dwarf bamboo (Sasa spp.) vegetation on the forest floor which inhibited tree regeneration. Mortality rate was density-dependent; dense stands had higher mortality than sparse stands. Density change rate (recruitment rate - mortality rate) was, therefore, determined by both the type of understorey vegetation and stem density. Survivor growth was high in stands with high stem density and basal area. Mortality was dependent on basal area and altitude. Net basal area increment (net growth) was dependent only on stem density with other factors that influenced survivor growth and mortality omitted. Tree size variability decreased in stands with high tree size variability whereas it increased in stands with low size variability. Based on the obtained models for density change rate and net basal area increment, trajectories of stands were illustrated on a log-log diagram of stem density and basal area. The predicted differences in trajectories as affected by the understorey vegetation type indicated the importance of dwarf bamboo vegetation for forest dynamics on Hokkaido.