Eiji Uheda

The growth and nitrogen fixation of Azolla filiculoides Lam. in polluted water

Abstract The growth and nitrogen fixation of Azolla filiculoides Lam. cultured in water of a polluted pond and two types of secondary effluent were investigated. The gradual decreases in the growth and acetylene reduction activity (ARA) of the plants in pond water were due mainly to a phosphorus deficiency. In secondary effluent, ammonium was an important factor limiting growth and ARA. The ARA was inhibited by ammonium in the medium even at 0.1 mM, whereas the growth rate in up to 1 mM ammonium was comparable with that of plants grown without ammonium. Nitrite and volatile organic acids in culture media were very toxic to Azolla plants. In secondary effluent containing nitrite, the plants shed their roots, followed by the fragmentation of fronds, and eventually died.

Response of six Azolla species to transient high-temperature stress

Abstract Response of eight Azolla strains from six species to transient exposure to high-temperature stress (above 40°C) was examined. Each Azolla strain showed differential tolerances to the stress. Based on growth and survival after the treatment, we concluded that the order of tolerance of six species to transient high-temperature stress was as follows: A. pinnata>A. microphylla, A. mexicana>A. caroliniana, A. filiculoides>A. rubra. Treatment with high-temperature stress also caused the rapid abscission of root and branches of Azolla. The temperature and time that caused rapid abscission depended on the species. In A. filiculoides and A. microphylla, the abscised branches were alive and proliferative when the temperature stress was short-term.

Immunolocalization and possible roles of pectins during pollen growth and callose plug formation in angiosperms.

To elucidate the possible roles of pectins during the growth of angiosperm pollen, we studied the distribution and changes in the properties of pectin in the pollen grains and tubes of Camellia japonica, Lilium longiflorum, and five other species at different growth stages by immunoelectron microscopy with monoclonal antibodies JIM5, against de-esterified pectin, and JIM7, against esterified pectin. We also studied the localization of arabinogalactan proteins, which are regarded as pectin-binding proteins, with monoclonal antibodies JIM13 and LM2, against arabinogalactan proteins. Similar results were obtained for all species: JIM5 labeled the intine and part of the callose layer in germinated pollen grains, and labeled the outer layer of the tube wall, the Golgi vesicles, and the callose plug in the pollen germinated in vitro, but did not label any part of immature pollen grains. In contrast, JIM7 labeled the intine of both immature and mature pollen grains, labeled the Golgi vesicles around the Golgi bodies, and strongly labeled the outer layer of the cell wall and the Golgi vesicles in the tube tip region. On the other hand, the distribution of arabinogalactan proteins detected with JIM13 was different for each species, and does not suggest a close relationship between pectin and arabinogalactan proteins. LM2 scarcely reacted with the specimens. We discuss the contribution of pectins to tube wall formation and fertilization and deduce a mechanism of callose plug formation.

Response of 19 Azolla strains to a high concentration of ammonium ions

The response of 19 Azolla strains from five species to 20 mM ammonium ions was examined. The response varied even among strains of the same species. The tolerance indexes of both relative growth rate and nitrogen fixation [(values for plants grown in the presence of ammonium ions)/(values for plants grown in the absence of ammonium ions)] showed that ammonium ions did not always simultaneously inhibit growth and nitrogen fixation of individual strains. The tolerance of each Azolla–Anabaena association to ammonium ions is assumed to be determined separately by both the host fern and the symbiotic Anabaena. The inhibitory effects of ammonium appeared predominantly in the mature region of Azolla–Anabaena associations. In the sensitive strains, both chlorophyll content and the number of cyanobionts were reduced only in the mature region when ammonium ions were present. Possible strains for treatment of wastewater, which contains a high concentration of ammonium ions, are discussed.

Differences in growth rate, nitrogen fixation and numbers of cyanobionts and heterocysts among three Azolla pinnata var. pinnata strains

The differences in nitrogen fixation, growth rate and numbers of cyanobionts and heterocysts among three Azolla pinnata var. pinnata strains were examined. The relative growth rate (RGR) and nitrogen fixation of PP7002 and PP7003 were significantly low compared with those of PP7005. The application of ammonium ions at 0.2 mM or more increased the growth rate of PP7002 and PP7003, but not PP7005. The numbers of cyanobionts and heterocysts in the mature region of PP7002 and PP7003 were statistically lower than those of PP7005. The low nitrogen-fixing activity of PP7002 and PP7003 as compared with PP7005 might be related to the restricted number of heterocysts. In PP7002 and PP7003, nitrogen fixation might be insufficient for full growth.

Aspects of the Very Rapid Abscission of Azolla Branches: Anatomy and Possible Mechanism

Sodium azide caused the rapid abscission of branches only in the mature regions of Azolla filiculoides. The abscission started at or after 5 min of exposure to 0.5 mM sodium azide. The presence of ethylene at 70 ppm neither induced rapid abscission within 3 h nor influenced the rapid abscission of Azolla branches caused by sodium azide. Cycloheximide or actinomycin D had no inhibitory effect on the abscission that was caused by sodium azide. Light and electron microscopy revealed that large, flattened cells, present at the base of each branch, formed an abscission layer. Sequential observations of large flattened cells from the apical to the mature region of Azolla indicated that the middle lamella between the cells in the mature region had already been partially degraded, even when a branch was still attached to a plant. Sodium azide induced the rapid dissolution of the small amount of middle lamella that remained between the large, flattened cells in the mature region, as well as modification of the primary wall of these cells. These events appeared to weaken both the cell walls and the adhesion between the cells, causing cell expansion and rounding. The expansion and rounding of the large, flattened cells facilitated the complete separation of these cells and the subsequent separation of the branch. Possible mechanisms for the rapid abscission of Azolla branches upon treatment with sodium azide are discussed.

Disappearance of Symbiotic Algae in the Azolla-Anabaena Association Subjected to Transitory Exposure to Ammonium

Abstract When Azolla japonica was grown on media containing ammonium at high concentrations, the ammonium levels within the Azolla tissue increased rapidly and reached the same levels as those in the media. When these Azolla fronds were transferred to a N-free medium, yellow spots were observed. In the crushed yellow spots, hardly any filaments of symbiotic algae could be detected under a microscope. Furthermore, the yellow spots cut from whole plants were able to regenerate to normal fronds only on a medium containing combined nitrogen at low concentrations. These regenerated fronds were free from Anabaena, did not exhibit acetylene reduction activity and failed to survive on a N-free medium.

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH.

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0-5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.

Breakdown of middle lamella pectin by ●OH during rapid abscission in Azolla

Azolla, a small water fern, abscises its roots and branches within 30 min upon treatment with various stresses. This study was conducted to test whether, in the rapid abscission that occurs in Azolla, breakdown of wall components of abscission zone cells by (●) OH is involved. Experimentally generated (●) OH caused the rapid separation of abscission zone cells from detached roots and the rapid shedding of roots from whole plants. Electron microscopic observations revealed that (●) OH rapidly and selectively dissolved a well-developed middle lamella between abscission zone cells and resultantly caused rapid cell separation and shedding. Treatment of abscission zones of Impatiens leaf petiole with (●) OH also accelerated the separation of abscission zone cells. However, compared with that of Azolla roots, accelerative effects in Impatiens were weak. A large amount of (●) OH was cytochemically detected in abscission zone cells both of Azolla roots and of Impatiens leaf petioles. These results suggest that (●) OH is involved in the cell separation process not only in the rapid abscission in Azolla but also in the abscission of Impatiens. However, for rapid abscission to occur, a well-developed middle lamella, a unique structure, which is sensitive to the attack of (●) OH, might be needed.

Analysis of apical hook formation in Alaska pea with a 3-D clinostat and agravitropic mutant ageotropum

The formation of the apical hook in dicotyledonous seedlings is believed to be effected by gravity in the dark. However, this notion is mostly based on experiments with the hook formed on the hypocotyl, and no detailed studies are available with the developmental manners of the hook, particularly of the epicotyl hook. The present study aims at clarifying the dynamics of hook formation including the possible involvement of gravity. Time-course studies with normal Alaska pea (Pisum sativum L., cv. Alaska) and an agravitropic pea mutant, ageotropum, under the 1-g conditions and on a 3-D clinostat revealed that (1) the apical hook of the epicotyl forms by the development of the arc-shaped plumule of the embryo existing in the non-germinated seed. The process of formation consists of two stages: development and partial opening, which are controlled by some intrinsic property of the plumule, but not gravity. Approximately when the epicotyl emerges from the seed coat, the hook is established in both pea varieties. In Alaska the established hook is sustained or enhanced by gravity, resulting in a delay of hook opening compared with on a clinostat, which might give an incorrect idea that gravity causes hook formation. (2) During the hook development and opening processes the original plumular arc holds its orientation unchanged to be an established hook, which, therefore, is at the same side of the epicotyl axis as the cotyledons. This is true for both Alaska and ageotropum under 1-g conditions as well as on the clinostat, supporting finding (1). (3) Application of auxin polar transport inhibitors, hydroxyfluorenecarboxylic acid, naphthylphthalamic acid, and triiodobenzoic acid, suppressed the curvature of hook by equal extents in Alaska as well as ageotropum, suggesting that the hook development involves auxin polar transport probably asymmetrically distributed across the plumular axis by some intrinsic property of the plumule not directly related with gravity action.