The extracts of Japanese willow tree species are effective forapoptotic desperation or differentiation of acute myeloid leukemia cells.

BACKGROUND: The antileukemic activity of hot water extract of plant parts of some Japanese willow tree species grown at different levels of nitrogen were examined. MATERIALS AND METHODS: Water extracts of willow leaves were prepared for this studies in different level of nitrogen nutrition. RESULTS: The extracts obtained from the leaves and stem exhibited anti-leukemic activities prominently. The crude hot water extracts of the young growing parts including apex, matured leaves and stem, killed the blasts of acute myeloid leukemia (AML) cells, (HL60 and NB4) after 48h incubation, however, such desperation was far less in the root extract. Similar to the plant parts, response of extracts obtained from different willow species was not identical; the proportion of dead cells relative to whole cells of the culture medium ranged from 21% to 93% among the species. Leaf extracts obtained from the responsive willow species decreased the live cell percentage and increased the dead cell percentage at higher level of nitrogen nutrition. The mode of desperation of leaf extract treated AML cells in such species appeared to be cell apoptosis as shown by binding with fluorescein isothiocyanate (FITC) -labeled Annexin V. CONCLUSION: Differentiation of alive AML cells continued unabated and apoptosis was poor when extract of an unresponsive species added to the culture medium.

Evaluation of salt tolerance in ectoine-transgenic tomato plants (Lycopersicon esculentum) in terms of photosynthesis, osmotic adjustment, and carbon partitioning.

Ectoine is a common compatible solute in halophilic bacteria. Its biosynthesis originates from L-aspartate ß-semialdehyde and requires three enzymes: L-2, 4-diaminobutyric acid aminotransferase (gene: ect B), L-2,4-diaminobutyric acid acetyl transferase (gene: ect A) and L-ectoine synthase (gene: ect C). Genetically engineered tomato plants expressing the three H. elongata genes (ectA, ectB, and ectC) generated showed no phenotypic abnormality. Expression of the ectoine biosynthetic genes was detected in the T3 transgenic plants by Northern blot analysis. The ectoine accumulating T3 plants were evaluated for salt tolerance by examining their photosynthestic activity, osmotic adjustment and carbon partitioning. Nuclear magnetic resonance (NMR) detected the accumulation of ectoine. The concentration of ectoine increased with increasing salinity. The transgenic lines showed higher activities of peroxidase, while the malondialdehyde (MDA) concentration was decreased under salinity stress condition. In addition, preservation of higher rates of photosynthesis and turgor values as compared to control was evident. Within a week of ( 13) CO 2 feeding, salt application led to increases in the partitioning of ( 13) C into roots at the expense of ( 13) C in the other plant parts. These results suggest that under saline conditions ectoine synthesis is promoted in the roots of transgenic plants, leading to an acceleration of sink activity for photosynthate in the roots. Subsequently, root function such as water uptake is improved, compared with wild-type plants. In this way, the photosynthetic rate is increased through enhancement of cell membrane stability in oxidative conditions under salt stress.

Potassium deficiency affects water status and photosynthetic rate of the vegetative sink in green house tomato prior to its effects on source activity.

The potassium requirement of green house tomatoes is very high for vegetative growth and fruit production. Potassium deficiency in plants takes long time for expression of visible symptoms. The objective of this study is to detect the deficiency early during the vegetative growth and define the roles of aquaporin and K-channel transporters in the process of regulation of water status and source-sink relationship. The tomato plants were grown hydroponically inside green house of Hiroshima University, Japan and subjected to different levels of K in the rooting medium. Potassium deficiency stress decreased photosynthesis, expansion and transport of ¹4C assimilates of the source leaf, but the effects became evident only after diameter expansion of the growing stem (sink) was down-regulated. The depression of stem diameter expansion is assumed to be associated with the suppression of water supply more than photosynthate supply to the organ. The stem diameter expansion is parameterized by root water uptake and leaf transpiration rates. The application of aquaporin inhibitor (AgNO3) decreased leaf water potential, stem expansion and root hydraulic conductance within minutes of application. Similar results were obtained for application of the K-channel inhibitors. These observations suggested a close relationship between stem diameter expansion and activities of aquaporins and K-channel transporters in roots. The deficiency of potassium might have reduced aquaporin activity, consequently suppressing root hydraulic conductance and water supply to the growing stem for diameter expansion and leaf for transpiration. We conclude that close coupling between aquaporins and K-channel transporters in water uptake of roots is responsible for regulation of stem diameter dynamics of green house tomato plants.

Effect of terminal locations of pods on biomass production and 13C partitioning in a fasciated stem soybean Shakujo.

This study addresses the hypothesis that stagnation of soybean yield on the farm can be improved by selection of a physiological trait favoring carbon assimilate partitioning to terminally placed pods versus genotypes having axillary pods at close plant spacing. (13)C was fed to source-sink units comprising a leaf, axillary/terminal pods, and petioles at upper and lower positions of the stem axis in two soybean cultivars, namely Shakujo and Enrei, at different densities of populations. The cultivars differ significantly in architecture, Shakujo bearing a few hundreds of pods in close succession to one another in a terminally placed raceme, in contrast to Enrei having axillary racemes. Pod yield per plant was higher in Enrei than in Shakujo at low density, but Shakujo out-yielded Enrei at close spacing. Population density decreased yield per plant and altered the pattern of assimilate partitioning significantly within the plants for both varieties. At high density more assimilates moved to the upper parts at the cost of the lower parts. The terminally placed pods of Shakujo were advantaged to receive assimilates under density stress. No benefit was accrued to pod filling of Enrei, however, under this condition.

High temperature effects on photosynthate partitioning and sugar metabolism during ear expansion in maize (Zea mays L.) genotypes.

Short hot and dry spells before, or during, silking have an inordinately large effect on maize (Zea mays L.; corn) grain yield. New high yielding genotypes could be developed if the mechanism of yield loss were more fully understood and new assays developed. The aim here was to determine the effects of high temperature (35/27 degrees C) compared to cooler (25/18 degrees C) temperatures (day/night). Stress was applied for a 14 d-period during reproductive stages prior to silking. Effects on whole plant biomass, ear development, photosynthesis and carbohydrate metabolism were measured in both dent and sweet corn genotypes. Results showed that the whole plant biomass was increased by the high temperature. However, the response varied among plant parts; in leaves and culms weights were slightly increased or stable; cob weights decreased; and other ear parts of dent corn also decreased by high temperature. Photosynthetic activity was not affected by the treatments. The (13)C export rate from an ear leaf was decreased by the high temperature treatment. The amount of (13)C partitioning to the ears decreased more than to other plant parts by the high temperature. Within the ear decreases were greatest in the cob than the shank within an ear. Sugar concentrations in both hemicellulose and cellulose fractions of cobs in sweet corn were decreased by high temperature, and the hemicellulose fraction in the shank also decreased. In dent corn there was no reduction of sugar concentration except in the in cellulose fraction, suggesting that synthesis of cell-wall components is impaired by high temperatures. The high temperature treatment promoted the growth of vegetative plant parts but reduced ear expansion, particularly suppression of cob extensibility by impairing hemicellulose and cellulose synthesis through reduction of photosynthate supply. Therefore, plant biomass production was enhanced and grain yield reduced by the high temperature treatment due to effects on sink activity rather than source activity. Heat resistant ear development can be targeted for genetic improvement.

Barium toxicity effects in soybean plants.

Barium (Ba)-induced phytotoxicity at 100, 1000, or 5000 microM Ba in soybean plants (Glycine max) was investigated under hydroponic culture conditions. Soybean growth and leaf photosynthetic activity were significantly inhibited by all three levels of Ba treatments. In the case of photosynthetic activity, 5000 microM Ba treatment shutdown stomatal opening and perturbed carbon fixation metabolism and translocation. However, 100 and 1000 microM Ba treatments shut down stomatal opening and inhibited carbon fixation, but without perturbation of leaf carbon fixation-related metabolism. Potassium (K) absorption by soybean roots was also reduced in all three Ba treatments. This decreased K absorption reduced K localization at guard cells. Barium accumulation in guard cells also inhibited K transport from epidermal cells to guard cells. This lack of K in guard cells resulted in stomatal closure. As a result of inhibition of K transport into guard cells and stomatal shutdown, photosynthetic activity and plant productivity were inhibited. Our experiment indicates that Ba has phytotoxic effects on soybean plants by inhibiting photosynthesis.

Willow leaves' extracts contain anti-tumor agents effective against three cell types.

Many higher plants contain novel metabolites with antimicrobial, antifungal and antiviral properties. However, in the developed world almost all clinically used chemotherapeutics have been produced by in vitro chemical synthesis. Exceptions, like taxol and vincristine, were structurally complex metabolites that were difficult to synthesize in vitro. Many non-natural, synthetic drugs cause severe side effects that were not acceptable except as treatments of last resort for terminal diseases such as cancer. The metabolites discovered in medicinal plants may avoid the side effect of synthetic drugs, because they must accumulate within living cells. The aim here was to test an aqueous extract from the young developing leaves of willow (Salix safsaf, Salicaceae) trees for activity against human carcinoma cells in vivo and in vitro. In vivo Ehrlich Ascites Carcinoma Cells (EACC) were injected into the intraperitoneal cavity of mice. The willow extract was fed via stomach tube. The (EACC) derived tumor growth was reduced by the willow extract and death was delayed (for 35 days). In vitro the willow extract could kill the majority (75%-80%) of abnormal cells among primary cells harvested from seven patients with acute lymphoblastic leukemia (ALL) and 13 with AML (acute myeloid leukemia). DNA fragmentation patterns within treated cells inferred targeted cell death by apoptosis had occurred. The metabolites within the willow extract may act as tumor inhibitors that promote apoptosis, cause DNA damage, and affect cell membranes and/or denature proteins.

Molecular control of gene co-suppression in transgenic soybean via particle bombardment.

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root.

To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism.

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO(2)) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from (15)N-labeled NO(2) taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen ( UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with (15)NO(2). Secondly, tobacco ( Nicotiana tabacum L.) plants fed with (15)N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen-oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.

Partitioning of 13C-labelled photosynthate varies with growth stage and panicle size in high-yielding rice.

A super-high-yielding rice (Oryza sativa L.) cultivar, Takanari, and a traditional japonica rice cultivar, Nakateshinsenbon, were grown under field conditions to compare partitioning of 13C-labelled photosynthate to different plant organs during the period of reproductive development. The flag leaf and the two leaves immediately below it on the main culm were exposed individually to 13CO2 and the movement of the heavy carbon isotope to grains, hull, panicle branches and vegetative parts of plant was assessed. Also, the effect of a reduction of sink size on the partitioning of 13C to different organs was studied by removing some of the primary branches of the panicle. 13C taken up by the three leaves in the post-heading period, moved mostly to the grains and hull of the panicle. At this stage, the uppermost three leaves and the panicle consisted of a single source-sink unit. Partitioning of 13C to the rest of the vegetative structures of the plant was minimal. In the case of Nakateshinsenbon, the flag leaf supplied most of the carbon assimilates for the grains and contributions from the other two leaves were much smaller. However, in Takanari, the contribution of 13C to grains from the second leaf was equivalent to that of the flag leaf. In Takanari, removal of more than one third of the primary branches of the panicle significantly reduced partitioning from the third leaf of the culm, but partitioning from the flag leaf was not significantly changed. In contrast, branch removal treatment significantly depressed transport of carbon assimilates from the flag leaf in Nakateshinsenbon. The obligatory nature of the source-sink relationship in rice is discussed. It is concluded that in lower-yielding traditional rice, photosynthesis in the flag leaf supplies carbon assimilates to the developing grains. But in the super-yielding rice Takanari, the main source area is extended to include the two leaves below the flag leaf so as to sustain an extra large panicle. Even greater grain-filling is possible in super-yielding rice, if the source area is increased further.

Role of exudation of organic acids and phosphate in aluminum tolerance of four tropical woody species.

Responses of Melaleuca leucadendra (L.) L., Melaleuca cajuputi Powell, Acacia auriculiformis A. Cunn. ex Benth. and Eucalyptus camaldulensis Dehnh. to aluminum (Al) toxicity at low pH are poorly understood. We investigated effects of low pH and exudation of ligands by roots on Al tolerance of these species. Seedlings were grown hydroponically in nutrient solutions at pH 4.2 or 3.5 containing AlCl3 at concentrations ranging from 0 to 4 mM Al. The presence of 4 mM Al at pH 3.5 depressed growth in all species. Growth depression was greatest in E. camaldulensis, least in A. auriculiformis. In the low Al treatment (0.5 mM Al), roots of M. cajuputi tended to have the highest Al concentration among species, whereas in the 4 mM Al treatment, the highest Al concentration was found in roots of E. camaldulensis. Aluminum application enhanced root exudation of citrate in all species, with the enhancement in M. cajuputi, M. leucadendra and A. auriculiformis being similar and much greater than in E. camaldulensis. Exudation of oxalate and phenolic compounds was greater in E. camaldulensis than in the other species. The presence of Al enhanced phosphate exudation in all species, particularly in A. auriculiformis. Acacia auriculiformis was tolerant to low pH, probably because the presence of an unknown substance increased the pH. Application of 0.38 mM Al alleviated the toxicity of the pH 3.5 treatment in E. camaldulensis and M. cajuputi, whereas low pH alleviated Al toxicity in A. auriculiformis. We conclude that exudation of ligands such as citrate and phosphate only partly accounts for interspecific differences in Al tolerance among the tropical woody plants studied, whereas the reciprocal alleviation of Al toxicity and low pH differed considerably among the species.

The effect of willow leaf extracts on human leukemic cells in vitro.

The young developing leaves of willow (Salix safsaf, Salicaceae) trees have antileukemic activity. After a 24-h incubation in vitro, the crude water extracts of the leaves killed a majority of the blasts of acute myeloid leukemia (AML, 73.8%).

Circadian rhythm of stem and fruit diameter dynamics of Japanese persimmon (Diospyrus kaki Thunb.) is affected by deficiency of water in saline environments.

Early diagnosis of water deficiency is essential to mitigate salt stress injury in plants. The effects of salt stress during the fruit growth stage on stem and fruit diameters of Japanese persimmon trees (Diospyrus kaki Thunb.) were measured by a micromorphometric technique under greenhouse conditions. This technique is less cumbersome and more precise in comparison to measurement of water potential in a small pressure chamber. The effect of stress was measured on photosynthetic rate, pre-dawn water potential, stomatal conductance, transpiration and Na+ and K+ contents of the stem and leaves. Salt stress was imposed by irrigating the plants with NaCl solution. Stem and fruit diameters of the plants given the control treatment started to decrease around 0600 h and reached a minimum at 1400 h. Salt stress did not change the diurnal pattern of response in stem and fruit diameter dynamics, but decreased the amplitude of the circadian rhythm by influencing both declining and recovery phases. The effect of salt stress on stem diameter appeared after 1 d of treatment, and on the third day in the fruit. Salt stress also reduced water potential, photosynthesis, transpiration and stomatal conductance, and increased concentrations of Na and K in the plant parts. Most of these effects were expressed after a lag period of 5 d of salt application. Utilization of micromorphometric techniques for early diagnosis of water deficiency in salt-prone environments is recommended based on results of this study.