Katsumi Kojima

Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro

The use of in vitro shoot cultures to evaluate osmotic and salt tolerance and the effects of salt and mannitol in the medium on proline and sugar accumulation were investigated in two poplar species, P. euphratica and P. alba cv. Pyramidalis × P. tomentosa. Shoot length, leaf number, whole plant dry weight, and the accumulation of proline and total soluble sugars in leaves were quantified after 2 weeks. All P.euphratica plantlets survived at all levels of mannitol and NaCl, while the mortality of P. alba cv. Pyramidalis × P. tomentosa increased both at the mannitol and the NaCl treatments. A significant increase in proline accumulation was observed in both young and mature P. euphratica leaves at 200 mM mannitol and above, and at 150 mM NaCl and above. The total soluble sugar content increased in young P. euphratica leaves at 250 mM NaCl; however, it decreased in the mature leaves. Similar increases of the total soluble sugar content were not seen in P. alba cv. Pyramidalis × P. tomentosa plants in response to either mannitol or NaCl treatment. Our results suggest that accumulated proline and sugars promote osmotic and salt tolerance. The effects of accumulated proline and total soluble sugars on leaves are discussed in relation to growth and osmotic adjustment.

Changes in carbon storage in fallow forests in the tropical lowlands of Borneo.

Abstract The range of CO 2 emissions to the atmosphere from tropical land-use change has long been debated. A major source of uncertainty is attributable to the fluxes of CO 2 due to changes in tropical land use. For example, estimate is different depending on whether the biomass or carbon content of tropical forests is totally lost by burning or recovered by regrowth. To estimate CO 2 absorption by fallow forests after shifting cultivation, we investigated the floristic composition and measured the biomass of fallow forests in East Kalimantan, Indonesia. Above-ground biomass of fallow forests were 8–10xa0t/ha at one-year-old stands and 45–56xa0t/ha at 10–12-year-old stands, regardless of the floristic composition. It seems reasonable to suppose that 7.4% of carbon released to the atmosphere by tropical rain forest destruction is reabsorbed and stored by fallow forests in Indonesia. If this is a universal trend in other tropical forests, a significant amount of CO 2 is reabsorbed in fallow forests. We consider that fallow forests like the one studied here are substantial carbon sinks and help to offset the effects of anthropogenic CO 2 emissions.

Aluminum distribution and reactive oxygen species accumulation in root tips of two Melaleuca trees differing in aluminum resistance

To elucidate the mechanism of the high aluminum (Al) resistance of a Myrtaceae tree, Melaleuca cajuputi Powell, we investigated the responses of root tips to Al and compared them with those of an Al-sensitive species, M. bracteata F. Muell. Roots of seedlings of both species were treated with a calcium solution (pH 4.0) containing 0 or 1 mM AlCl3. After 3 h of Al treatment, inhibition of root elongation and deposition of callose and lignin in root tips, typical signs of Al injury, were induced in M. bracteata but not in M. cajuputi, yet Al accumulation in root tips was similar in both species. These results indicate that internal Al tolerance mechanisms, not Al exclusion mechanisms, are responsible for the Al resistance of M. cajuputi. After 3 h of Al treatment, amount of Al tightly bound to root tips, Al remaining after washing with a desorbing solution, was less in M. cajuputi than in M. bracteata. In M. bracteata, 6 h of Al treatment triggered the accumulation of hydrogen peroxide (H2O2) in root tips despite the upregulation of antioxidant mechanisms, activity of peroxidase and concentration of reduced glutathione. In M. cajuputi, 6 h of Al treatment did not affect the concentration of H2O2, but decreased activity of peroxidase, and increased concentration of reduced glutathione in root tips. These results suggest that the less Al tightly bound to root tips is involved in the suppressing the H2O2 accumulation and the internal Al tolerance in M. cajuputi, and that the H2O2 accumulation or changes in cellular environment that bring about H2O2 accumulation despite the upregulation of antioxidant mechanisms results in Al-induced inhibition of root elongation in M. bracteata.

Aluminum tolerance and aluminum-induced deposition of callose and lignin in the root tips of Melaleuca and Eucalyptus species

We exposed the roots of nine Myrtaceae species (Melaleuca bracteata F. Muell., Melaleuca cajuputi Powell, Melaleuca glomerata F. Muell., Melaleuca leucadendra [L.] L., Melaleuca quinquenervia [Cav.] S.T. Blake, Melaleuca viridiflora Sol. ex Gaertner, Eucalyptus camaldulensis Dehnh., Eucalyptus deglupta Bl., and Eucalyptus grandis W. Hill ex Maiden) to 1u2009mM Al in a nutrient solution for either 24u2009h or 20 days to evaluate their sensitivity to excess Al and to determine whether callose and lignin formation can be used as interspecific indicators of Al sensitivity. Inhibition of root elongation by Al varied among the species. Melaleuca leucadendra, M. cajuputi, E. grandis, M. quinquenervia, and E. deglupta were tolerant to 1u2009mM Al, whereas M. viridiflora, E. camaldulensis, M. glomerata, and M. bracteata were sensitive to 1u2009mM Al. We found that Al induced callose formation in the root tips of each species, but lignin was formed only in the root tips of the most sensitive species, M. bracteata. Root elongation at 1u2009mM Al was negatively correlated with Al-induced callose formation but not with the Al-induced lignin formation in the root tips. These results suggest that Al-induced callose formation, rather than lignin formation, can be used as an interspecific indicator of Al sensitivity.

Role of aluminum-binding ligands in aluminum resistance of Eucalyptus camaldulensis and Melaleuca cajuputi

We investigated the roles of Al-binding ligands in Al exclusion from roots and in internal Al detoxification in roots as Al resistance mechanisms in two Al-resistant Myrtaceae trees, Eucalyptus camaldulensis Dehnh. and Melaleuca cajuputi Powell. The amounts of ligands secreted from roots and contained in root tips of these species were compared with those of an Al-sensitive species, Melaleuca bracteata F. Muell., after the roots were exposed to 0 or 1xa0mM AlCl3 solution. Secretion of well-known ligands (citrate, oxalate, and malate) from roots under Al treatment was low in all species. However, in E. camaldulensis, the Al-binding capacity of root exudates under Al treatment was considerable and was higher than that in M. bracteata. Gel filtration chromatography revealed that a low-molecular-weight Al-binding ligand was secreted from roots in response to Al only in E. camaldulensis. On the other hand, the Al-binding capacity of cell sap in root tips under Al treatment was similar for the resistant and sensitive species. These results suggest that Al exclusion by secretion of the unknown low-molecular-weight Al-binding ligand from roots contributes to the Al resistance of E. camaldulensis, whereas M. cajuputi has developed Al-resistance mechanisms other than secretion of ligands from roots or concentration of internal ligands in root tips.

Effects of flooding on downstream processes of glycolysis and fermentation in roots of Melaleuca cajuputi seedlings

We investigated the energy metabolism in roots of flooded Melaleuca cajuputi Powell, a tropical flood-tolerant tree species, by measuring adenylate concentrations and activities of glycolytic and fermentative enzymes under flooded conditions. Adenylate energy charge (AEC) decreased slightly to 0.72 on the second day of flooding and recovered to around 0.8 by the fourth day of flooding. Activities of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) increased initially and then decreased to the control level after 14 days of flooding. On the other hand, activities of pyruvate kinase (EC 2.7.1.40), phosphoenolpyruvate phosphatase (EC 3.1.3.2), and a series of phosphoenolpyruvate carboxylase (EC 4.1.1.31), malate dehydrogenase (EC 1.1.1.37), and NADP dependent malic enzyme (EC 1.1.1.40), which can convert PEP into pyruvate, were not induced in flooded roots throughout the experiment. These results suggest that neither the downstream reactions of glycolysis nor ATP production via glycolysis was enhanced by flooding, whereas alcohol fermentation was enhanced. With the low ATP yield of the glycolysis–alcohol fermentation pathway and no induction of glycolytic enzymes, the glycolysis–alcohol fermentation pathway itself contributes little to ATP production in flooded roots of M. cajuputi. These physiological responses of M. cajuputi to flooding may have the advantages of surviving flooded conditions because they can avoid exhaustion of sugar and accumulation of ethanol, a toxic end product of alcohol fermentation.

Structure of the pine (Pinus thunbergii) chlorophyll a/b-binding protein gene expressed in the absence of light

A gene for chlorophyll a/b-binding protein (cab) of pine (Pinus thunbergii) was isolated and sequenced. The gene (cab-6) contains an intron at a position equivalent to the type II cab genes of angiosperms. Transcript mapping analyses show that the amount of the mRNA in the dark is about half of that in the light. The cab-6 gene is expressed in dark-grown seedlings at a very high level, differing from angiosperm cab genes which are induced by light. The cab-6 gene typifies the coniferous plant cab genes in light-independent gene expression.

Ectomycorrhizal Association Enhances Al Tolerance by Inducing Citrate Secretion in Pinus densiflora

To investigate the effects of ectomycorrhizal association on the aluminum (Al) tolerance of Pinus densiflora Sieb. & Zucc., seedlings with or without ectomycorrhizal association with the fungus Pisolithus tinctorius (Pers.) Coker & Couch were exposed to 1 mM Al. Association with P. tinctorius alleviated Al-induced inhibition of root elongation and biomass growth in the mycorrhizal seedlings. Secretion of malate and citrate, both low-molecular-weight organic acids that could detoxify Al by the formation of stable complexes, was investigated in P. tinctorius mycelia and in pine roots with and without P. tinctorius association. Citrate secretion from the P. tinctorius mycelia in vitro was stimulated by Al. Citrate secretion from the roots of the ectomycorrhizal seedlings was also stimulated by Al, but was not detected in the non-mycorrhizal seedlings. These results suggest that citrate secreted from the roots of the ectomycorrhizal seedlings was produced in the hyphae of P. tinctorius. Citrate secretion may play a role in enhancing the Al tolerance of host seedlings.

Identification of a Hydrolyzable Tannin, Oenothein B, as an Aluminum-Detoxifying Ligand in a Highly Aluminum-Resistant Tree, Eucalyptus camaldulensis

A hydrolyzable tannin isolated from roots of an aluminum-resistant eucalypt species can form nonphytotoxic complexes with aluminum ions. Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.

Analysis of clonal structure of Melaleuca cajuputi (Myrtaceae) at a barren sandy site in Thailand using microsatellite polymorphism

Abstract. The clonal structure of Melaleuca cajuputi growing in Thailand was investigated using ten polymorphic microsatellite markers and excavation of root connections. A large-scale test plot (25×45xa0m) and a small-scale one (10×12xa0m) within it were established in an M. cajuputi population at a barren sandy location in Narathiwat, southern Thailand (6° 30′ N, 101° 45′ E). Eleven clonal groups were identified from 31 ramets in the small-scale test plot based on root connections between ramets. To evaluate the effectiveness of microsatellite markers for genet analysis of M. cajuputi, clonal groups in the small-scale test plot identified by root connection were compared with genets determined by analysis of ten microsatellite markers. Most of the clonal groups identified by root connection completely matched the genets determined by the microsatellite markers. This indicates that microsatellite markers are very useful for genet analysis of M. cajuputi. Eleven genets were identified from 53 ramets in the large- and small-scale test plots using the ten microsatellite markers; the largest genet was about 23xa0m wide. This suggests that vegetative reproduction, i.e., shoot sprouting from roots, plays an important role in the establishment of M. cajuputi populations at this barren sandy site.