Expression of a fungal cyanamide hydratase in transgenic soybean detoxifies cyanamide in tissue culture and in planta to provide cyanamide resistance

Embryogenic tissue cultures of soybean were transformed by particle bombardment with a vector pCHZ-II that carries the coding sequence for cyanamide hydratase (Cah), an enzyme that converts toxic cyanamide to urea, from the soil fungus Myrothecium verrucaria. The Cah gene was driven by the constitutive Arabidopsis thaliana actin-2 promoter and terminated with its cognate terminator. This vector also carries the hygromycin phosphotransferase gene (hpt) driven by the potato (Solanum tuberosum) ubiquitin-3 promoter. Twelve individual lines of transgenic plants that were obtained under hygromycin selection expressed Cah mRNA and exhibited resistance to hygromycin in leaf tissue culture, while the untransformed tissues were sensitive. Cah enzyme activity was present in extracts of transformed leaves and embryogenic tissue cultures when measured by a colorimetric assay and the presence of the Cah protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Cah expression detoxified cyanamide in leaf callus and embryogenic cultures as well as in whole plants as shown by cyanamide resistance. The Cah-expressing plants grew and set seeds normally indicating that the Cah enzyme activity did not affect soybean plant metabolism. We also describe a test whereby callus was formed on cultured leaf tissue in the presence of hygromycin or cyanamide only if the hpt or Cah gene was expressed, respectively. This test is a convenient and cost-effective way to follow the marker gene in the primary regenerated plants and subsequent generations, which is particularly reliable for the hpt gene expression using hygromycin.     

Partitioning of 14C-Photosynthate of Leaves in Roots,Rhizome, and in Essential Oil and Curcumin in Turmeric (Curcuma longa L.)

Incorporation of photosynthetically fixed ¹⁴C was studied at different time intervals of 12, 24, and 36 h in various plant parts—leaf 1 to 4 from apex, roots, and rhizome—into primary metabolites—sugars, amino acids, and organic acids, and secondary metabolites—essential oil and curcumin—in turmeric. The youngest leaves were most active in fixing ¹⁴C at 24 h. Fixation capacity into primary metabolites decreased with leaf position and time. The primary metabolite levels in leaves were maximal in sugars and organic acids and lowest in amino acids. Roots as well as rhizome received maximum photoassimilate from leaves at 24 h; this declined with time. The maximum metabolite concentrations in the roots and rhizome were high in sugars and organic acids and least in amino acids. ¹⁴C incorporation into oil in leaf and into curcumin in rhizome was maximal at 24 h and declined with time. These studies highlight importance of time-dependent translocation of ¹⁴C-primary metabolites from leaves to roots and rhizome and their subsequent biosynthesis into secondary metabolite, curcumin, in rhizome. This might be one of factors regulating the secondary metabolite accumulation and rhizome development.     

A unique pantoyllactone glycoside system is activated in rice seedlings developing aerobically in the dark

Rice ( Oryza sativa L.) seedlings developing aerobically in the dark accumulated in the expanding coleoptile millimolar amounts of pantoyllactone glucoside (PLG) together with sugars and amino acids. Following leaf emergence and seedling development, the transformation of PLG into pantoyllactone primeveroside (PLP) was initiated inside the coleoptile, whose senescence was suspended in spite of the exhaustion of seed reserves and cessation of shoot growth. PLG, PLP and most of the sugars and amino acids remained inside the coleoptile. Light administration triggered shoot greening and coleoptile senescence that was accompanied by the conversion of PLG into the shoot-translocatable species PLP and by the utilization of the coleoptile-stored metabolites. Light-induced retrieval of amino acids and sugars from the coleoptile was 90% complete within 24 h while translocation of pantoyllactone glycosides started only 24–48 h after light administration. Conversely, no accumulation of PLG and PLP was detected in seedlings germinated and grown either aerobically in the light or in the dark under hypoxic conditions. The possible significance of the activation of the PLG-PLP system under specific environmental conditions is discussed.     

The distribution of organic and mineral components in leaves and stems of Ranunculus fluitans Lam.

The differences in chemical composition of leaves and stems of Ranunculus fluitans Lam. were investigated. Typical distribution of organic and inorganic compounds were generally found not to be influenced by factors as day-time, eutrophication, or age of the weed bed. Starch, sugars, amino acids, and organic acids were at a higher level in the stem, but inorganic cations and anions were accumulated in the leaf. The distribution pattern and the relative contents of individual sugars, acids, and inorganic ions are discussed in detail.     

The epidermis of barley leaves is a dynamic intermediary storage compartment of carbohydrates, amino acids and nitrate

Sugars, amino acids and nitrate were measured in the epidermis of barley seedlings and compared with whole leaf levels. I. Under all conditions, concentrations of glucose and fructose were lower in the epidermis than in the remaining leaf tissues; levels were lowest at the end of the dark period (0.2–1.4 m M ) and increased under conditions of inhibited assimilate export (3–6 m M ). 2. Epidermal sucrose concentrations were very low (« 0.2 m M ) even in excised leaves which had accumulated assimilates. 3. Similar to the sugars, amino acids were also less concentrated in the epidermis than in whole leaf extracts. However, the amino acid profiles showed cell type-specific differences. 4. Nitrate was accumulated in the epidermis. The epidermal pool decreased during nitrate starvation. Since no nitrate reductase activity was associated with the epidermis, nitrate was mobilised from the epidermis and metabolized presumably in the mesophyll. These results suggest that the epidermis functions as a regulated intermediary storage compartment of the leaves and that stored substances are readily available for remobilisation.     

Water Stress Induced Changes in the Amounts of Some Photosynthetic Assimilation Products and Respiratory Metabolites of Sunflower Leaves

Sunflower leaves, water-stressed under controlled conditions,contained greater amounts of amino acids as their water potentialdecreased, with glycine, serine, and glutamate increasing morethan alanine and aspartate. Proline accumulated only at severestress. Low O₂ concentration altered the amounts of amino acids,principally decreasing the amount of glycine and increasingserine. The changes in total pool size are related to previousresults on the accumulation of 14C and the specific activityof products. Photorespiration was large under water stress,where leaves accumulated carbon in glycine of low specific activity,and in 21% O₂, where both total amount and specific activityof glycine was greater than in 1.5% O₂. This suggests that thereare two pools of glycine, one controlled by O₂ and closely relatedto photosynthesis, the other non-photosynthetic and affectedby water stress. The organic acids suocinate, citrate, and fumarate increasedat small leaf-water potentials. Sucrose decreased in amountwith stress and was absent at the most severe stress; therewas less glucose and fructose. The amount of carbon lost fromsugars was similar to the amount accumulated in amino acidstogether with the carbon lost in respiration. It is concluded that stress decreased the flux of carbon fromphotosynthesis for the synthesis of amino acids and sugars butmore carbon from stored materials, principally sucrose, wasused in the production of organic acids and amino acids.     

Metabolite Profiling of Cheonggukjang,a Fermented Soybean Paste,Inoculated with Various Bacillus Strains during Fermentation

Metabolite profiling of Cheonggukjang inoculated with different Bacillus strains including Bacillus amyloliqueciens CH86-1, Bacillus licheniformis 58, and Bacillus licheniformis 67 during fermentation, was performed using gas chromatography-time of flight-mass spectrometry after derivatization, combined with multivariate statistical analysis. A total of 20 amino acids, 10 sugars, five sugar alcohols, and seven organic acids were identified in three Cheonggukjang samples. With fermentation time, most of the amino acids showed increasing amounts. On the other hand, most of the sugars including sucrose, fructose, and glucose decreasing patterns, and the amounts of organic acids varied. In order to observe differences in metabolites with fermentation time and inoculated Bacillus strains, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were carried out, respectively. On PCA plots, some sugars and organic acids including sucrose, fructose, glucose, mannose, succinic acid, and malonic acid, as well as most of the amino acids, contributed mainly to differentiation of the Cheonggukjang samples fermentation time. On the other hand, on PLS-DA, mannose, xylose, glutamic acid, and proline were mainly responsible for differentiating the Cheonggukjang among into various inoculated strains.     

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.     

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.     

Patterns of Assimilate Production and Translocation in Muskmelon (Cucumis melo L.) : I. Diurnal Patterns

Continuous monitoring of steady-state carbon dioxide exchange rates in mature muskmelon (Cucumis melo L.) leaves showed diurnal patterns of photosynthesis and respiration that were translated into distinct patterns of accumulation and phloem export of soluble sugars and amino acids. Leaf soluble sugar patterns in general followed the pattern of photosynthetic activity observed in the leaf, whereas starch accumulated steadily throughout the light period. Sugar and starch levels declined through the dark phase. Phloem exudate analysis revealed that diurnal levels of the major transport sugars (stachyose and sucrose) in the phloem did not appear to correlate directly with the photosynthetic activity of the leaf but instead were inversely correlated with leaf starch accumulation and degradation. The amino acid pool in leaf tissues remained constant throughout the diurnal period; however, the relative contribution of individual amino acids to the total pool varied with the diurnal photosynthetic and respiratory activity of the leaf. In contrast, the phloem sap amino acid pool size was substantially larger in the light than in the dark, a result primarily due to enhanced export of glutamine, glutamate, and citrulline during the light period. The results indicate that the sugar and amino acid composition of cucurbit phloem sap is not constant but varies throughout the diurnal cycle in response to the metabolic activities of the source leaf.     

Changes in Nonstructural Carbohydrates in Different Parts of Soybean (Glycine max [L.] Merr.) Plants during a Light/Dark Cycle and in Extended Darkness

Diurnal patterns of nonstructural carbohydrate (starch, sucrose, and hexose sugars) concentration were characterized in different parts (leaves, petioles, stems, and roots) of vegetative soybean (Glycine max [L.] Merr.) plants. Pronounced changes in all carbohydrate pools were observed in all plant parts during the normal photosynthetic period; however, starch accumulation within leaves accounted for more than 80% of the nonstructural carbohydrate accumulated by the plant during the light period. Efficiency of utilization of starch and sucrose during the normal dark period differed among organs, with leaves being most efficient in mobilizing starch reserves and roots being most efficient in utilizing sucrose reserves. The vast majority (about 85%) of the whole plant carbohydrate reserves present at the end of the photosynthetic period were utilized during the normal dark period. Sink leaf expansion ceased in plants transferred to extended darkness and the cessation in leaf expansion corresponded with carbohydrate depletion in the subtending source leaf and the remainder of the plant. Collectively, the results indicated that under the conditions employed, leaves are the whole plant's primary source of carbon at night as well as during the day.     

Contribution of inorganic components to osmotic adjustment and leaf folding for drought tolerance in pearl millet

Pearl millet, Pennisetum glaucum , is capable of adapting to severely dry environmental conditions. In order to elucidate the mechanism of adaptation to highly dehydrated conditions, we selected both tolerant (IP8210) and susceptible (IP8949) accessions from a total of 15 pearl millet accessions and characterized their morphological and physiological responses to severe drought stress. When these selected accessions were stressed with a severe drought treatment, the leaves of IP8210 exhibited upright folding, a response that effectively reduces the evaporative surface area of the canopy. On the contrary, the leaves of IP8949 exhibited wilting and did not appear to adapt to the drought stress. In comparison with IP8949, the capacity of osmotic adjustment (OA) was greater in both younger leaves and stems of IP8210, while their decrease in relative water content was different. IP8210 accumulated higher concentrations of NO₃^– than IP8949 in response to drought stress. In addition to inorganic solutes, several organic components such as sucrose, glucose, quaternary ammonium compounds, and amino acids including proline were also accumulated. IP8210 tended to accumulate more amino acids, typically due to the accumulation of asparagine and proline, while IP8949 accumulated more soluble sugars. While it is possible that K⁺ and NO₃^– were the major components contributing to osmotic regulations, sugars and amino acids might also function as a cytoprotectant, in addition to their role as osmoprotectants. Collectively, these results demonstrate that the morphological adaptation of leaf folding, OA in both the younger leaves and the stem, and the accumulation of NO₃^– and amino acids during earlier stress period contribute to superior drought tolerance that was exhibited in IP8210 of pearl millet.     

Influence of sequential prolonged periods of dark and light on pinitol concentration in clover and soybean tissue

The influence of prolonged dark periods on the concentration of pinitol in white clover ( Trifolium repens L. ev. Regal Ladino) and soybean [ Glycine max (L.) Merr.] tissue was investigated. Clover and soybean plants were harvested periodically throughout continuous dark periods of 40 and 44 h, respectively, followed by 24 h of continuous light. The foliage and root tissue of clover and foliage tissue of soybeans were analyzed for the concentration of glucose, fructose, sucrose and pinitol. Pinitol concentration (mg/g dry weight tissue) was unchanged by the prolonged dark period followed by 24 h of continuous light compared to tissue of plants harvested at 0-h and plants receiving normal photoperiods. Concentrations of the carbohydrates, in the tissue were depleted by the prolonged dark period. However after 6 h of light, the concentration of these sugars increased to concentrations equal to 0-h harvested plant tissue and tissue of plants receiving normal photoperiods. These results indicate that pinitol probably does not function as a source of reserve energy comparable to sucrose, glucose, and fructose.     

Inhibitory effect of the extract from Zizyphus jujuba leaves on sweet taste responses of the chorda tympani in the rat and hamster

1. One of the fractions obtained from the extract of Zizyphus jujuba leaves suppressed the response of the chorda tympani to sucrose, both in the rat and hamster. 2. In the rat and man, suppressive effect was found to be significant in responses to various sugars and artificial sweeteners but not in some sweet amino acids.     

Acifluorfen-induced isoflavonoids and enzymes of their biosynthesis in mature soybean leaves : whole leaf and mesophyll responses

Mature soybean (Glycine max L. cv Harosoy 63) leaves normally contain kaempferol-3-glycosides but they accumulate no other flavonoids. Whole leaves sprayed with the diphenyl ether herbicide Acifluorfen and maintained in the light developed small necrotic lesions and accumulated isoflavone aglycones, isoflavone glucosides, and pterocarpans. Isoflavonoid accumulation was preceded by induced activity for chalcone synthase (CHS) and by increased activity for phenylalanine ammonia-lyase (PAL) and UDP-glucose:isoflavone 7-O-glucosyl transferase (IGT). PAL and CHS activity was highest between 24 and 30 hours after treatment, isoflavone aglycones and pterocarpans at 48 hours, IGT at 72 hours, and isoflavone glucosides at 96 hours.