Circumnutation and distribution of phytohormones in <Emphasis Type="Italic">Vigna angularis</Emphasis> epicotyls

Circumnutation is a plant growth movement in which the tips of axial organs draw a circular orbit. Although it has been studied since the nineteenth century, its mechanism and significance are still unclear. Greened adzuki bean (Vigna angularis) epicotyls exhibited a clockwise circumnutation in the top view with a constant period of 60 min under continuous white light. The bending zone of circumnutation on the epicotyls was always located in the region 1–3 cm below the tip, and its basal end was almost identical to the apical end of the region where the epicotyl had completely elongated. Therefore, epidermal cells that construct the bending zone are constantly turning over with their elongation growth. Since exogenously applied auxin transport inhibitors and indole-3-acetic acid (IAA) impaired circumnutation without any effect on the elongation rate of epicotyls, we attempted to identify the distribution pattern of endogenous auxin. Taking advantage of its large size, we separated the bending zone of epicotyls into two halves along the longitudinal axis, either convex/concave pairs in the plane of curvature of circumnutation or pre-convex/pre-concave pairs perpendicular to the plane. By liquid chromatography–mass spectrometry, we found, for the first time, that IAA and gibberellin A₁ were asymmetrically distributed in the pre-convex part in the region 1–2 cm below the tip. This region of epicotyl sections exhibited the highest responsiveness to exogenously applied hormones, and the latent period between the hormone application and the detection of a significant enhancement in elongation was 15 min. Our results suggest that circumnutation in adzuki bean epicotyls with a 60 min period is maintained by differential growth in the bending zone, which reflects the hormonal status 15 min before and which is shifting sequentially in a circumferential direction. Cortical microtubules do not seem to be involved in this regulation.     

Genetic typing of the Senescence-Accelerated Mouse (SAM) strains with microsatellite markers

The Senescence-Accelerated Mouse (SAM) strains constitute a murine model of accelerated senescence originating from the ancestral AKR/J strains and consist of nine senescence-prone (SAMP) strains and four senescence-resistant (SAMR) strains. The chromosomes (Chrs) of the SAM strains were typed with 581 microsatellite markers amplified by PCR, and the fundamental genetic information of the SAM strains was obtained. One-third of the examined markers displayed polymorphism among the strains, and only two alleles were detected in almost all loci among the SAM and AKR/J strains. However, in 12 loci (5.6% of total 215 polymorphic markers), the third allele was detected among the SAM strains. The genetic typing and developmental history suggested that the SAM strains were related inbred strains developed by the accidental crossing between the AKR/J strain and other unknown strain(s). Comparison of the distribution of the loci in the SAMP and the SAMR series revealed notable differences in the four regions on Chrs 4, 14, 16, and 17. This indicated that some of these chromosomal sites might contain the genes responsible for accelerated senescence in the SAMP series. Received: 17 July 1998 / Accepted: 17 November 1998     

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.     

Genomic Evidence for Speciation with Gene Flow in Broadcast Spawning Marine Invertebrates

How early stages of speciation in free-spawning marine invertebrates proceed is poorly understood. The Western Pacific abalones, Haliotis discus, H. madaka, and H. gigantea, occur in sympatry with shared breeding season and are capable of producing viable F₁ hybrids in spite of being ecologically differentiated. Population genomic analyses revealed that although the three species are genetically distinct, there is evidence for historical and ongoing gene flow among these species. Evidence from demographic modeling suggests that reproductive isolation among the three species started to build in allopatry and has proceeded with gene flow, possibly driven by ecological selection. We identified 27 differentiation islands between the closely related H. discus and H. madaka characterized by high F_ST and d_A, but not high d_XY values, as well as high genetic diversity in one H. madaka population. These genomic signatures suggest differentiation driven by recent ecological divergent selection in presence of gene flow outside of the genomic islands of differentiation. The differentiation islands showed low polymorphism in H. gigantea, and both high F_ST, d_XY, and d_A values between H. discus and H. gigantea, as well as between H. madaka and H. gigantea. Collectively, the Western Pacific abalones appear to occupy the early stages speciation continuum, and the differentiation islands associated with ecological divergence among the abalones do not appear to have acted as barrier loci to gene flow in the younger divergences but appear to do so in older divergences.     

Biochemical characterization of thermostable β-1,4-mannanase belonging to the glycoside hydrolase family 134 from Aspergillus oryzae

Applied Microbiology and Biotechnology - A β-1,4-mannanase, termed AoMan134A, that belongs to the GH 134 family was identified in the filamentous fungus Aspergillus oryzae. Recombinant...     

Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.     

Aerobic exercise training reduces plasma endothelin-1 concentration in older women.

Endothelial function deteriorates with aging. On the other hand, exercise training improves the function of vascular endothelial cells. Endothelin-1 (ET-1), which is produced by vascular endothelial cells, has potent constrictor and proliferative activity in vascular smooth muscle cells and, therefore, has been implicated in regulation of vascular tonus and progression of atherosclerosis. We previously reported significantly higher plasma ET-1 concentration in middle-aged than in young humans, and recently we showed that plasma ET-1 concentration was significantly decreased by aerobic exercise training in healthy young humans. We hypothesized that plasma ET-1 concentration increases with age, even in healthy adults, and that lifestyle modification (i.e., exercise) can reduce plasma ET-1 concentration in previously sedentary older adults. We measured plasma ET-1 concentration in healthy young women (21-28 yr old), healthy middle-aged women (31-47 yr old), and healthy older women (61-69 yr old). The plasma level of ET-1 significantly increased with aging (1.02 +/- 0.08, 1.33 +/- 0.11, and 2.90 +/- 0.20 pg/ml in young, middle-aged, and older women, respectively). Thus plasma ET-1 concentration was markedly higher in healthy older women than in healthy young or middle-aged women (by approximately 3- and 2-fold, respectively). In healthy older women, we also measured plasma ET-1 concentration after 3 mo of aerobic exercise (cycling on a leg ergometer at 80% of ventilatory threshold for 30 min, 5 days/wk). Regular exercise significantly decreased plasma ET-1 concentration in the healthy older women (2.22 +/- 0.16 pg/ml, P < 0.01) and also significantly reduced their blood pressure. The present study suggests that regular aerobic-endurance exercise reduces plasma ET-1 concentration in older humans, and this reduction in plasma ET-1 concentration may have beneficial effects on the cardiovascular system (i.e., prevention of progression of hypertension and/or atherosclerosis by endogenous ET-1).     

Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414–12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2‐DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans.     

GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.     

The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis

SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.