Formation of N-acetylglutamate by extracts of higher plants

The enzymic synthesis of N-acetylglutamate was studied in extracts of higher plant tissues, especially in sugar beet leaves (Beta vulgaris L.). Sugar beet leaves had an enzyme that transferred the acetyl group either from acetyl-CoA or from N²-acetylornithine to glutamate. The enzyme was so unstable that special precautions were necessary for its detection and appreciable purification was impossible. The Km values were 2.5 and 0.025 mM for acetyl-CoA and N²-acetylornithine, respectively. The Km for glutamate was 23 mM with acetylornithine-glutamate transacetylase and 2.7 mM with acetyl-CoA-glutamate transacetylase. The pH optimum for acetyl-CoA-glutamate transacetylase was about 7.2 whereas that for acetylornithine-glutamate transacetylase was about 8.3. Acetylphosphate, N²-acetyl-2,4-diaminobutyrate, propionyl-CoA, and succinyl-CoA were not substrates.     

Kinetics of the Accumulation of Jasmonic Acid and Its Derivatives in Systemic Leaves of Tobacco (Nicotiana tabacum cv. Xanthi nc) and Translocation of Deuterium-Labeled Jasmonic Acid from the Wounding Site to the Systemic Site

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.     

Biosynthesis and defensive function of Nδ-acetylornithine, a jasmonate-induced Arabidopsis metabolite

Since research on plant interactions with herbivores and pathogens is often constrained by the analysis of already known compounds, there is a need to identify new defense-related plant metabolites. The uncommon nonprotein amino acid N(δ)-acetylornithine was discovered in a targeted search for Arabidopsis thaliana metabolites that are strongly induced by the phytohormone methyl jasmonate (MeJA). Stable isotope labeling experiments show that, after MeJA elicitation, Arg, Pro, and Glu are converted to Orn, which is acetylated by NATA1 to produce N(δ)-acetylornithine. MeJA-induced N(δ)-acetylornithine accumulation occurs in all tested Arabidopsis accessions, other Arabidopsis species, Capsella rubella, and Boechera stricta, but not in less closely related Brassicaceae. Both insect feeding and Pseudomonas syringae infection increase NATA1 expression and N(δ)-acetylornithine accumulation. NATA1 transient expression in Nicotiana tabacum and the addition of N(δ)-acetylornithine to an artificial diet both decrease Myzus persicae (green peach aphid) reproduction, suggesting a direct toxic or deterrent effect. However, since broad metabolic changes that are induced by MeJA in wild-type Arabidopsis are attenuated in a nata1 mutant strain, there may also be indirect effects on herbivores and pathogens. In the case of P. syringae, growth on a nata1 mutant is reduced compared with wild-type Arabidopsis, but growth in vitro is unaffected by N(δ)-acetylornithine addition.     

Characterization of three homoeologous cDNAs encoding chloroplast-targeted aminolevulinic acid dehydratase in common wheat

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid (ALA) is metabolized by ALA dehydratase (ALAD). Here, we isolated ALAD1 cDNA from common wheat (Triticum aestivum L.) and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 (TaALAD1) were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP (green fluorescent protein) fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations.     

DREB类转录因子介导的烟草抗非生物胁迫特性研究

检测并分析了转BnDREB1-5基因烟草叶片的持水性能,上、下表皮气孔大小和密度及叶绿素含量,并在自然失水7 h后检测了细胞的离子泄漏状况。结果表明:转基因烟草叶片单位时间内每平方厘米的失水量是野生型烟草叶片的62%;上表皮的气孔大于野生型,而气孔开度小于野生型;野生型烟草叶片上的气孔密度接近转基因烟草的1.5倍;野生型烟草叶片的叶绿素含量比转基因烟草叶片高29%;野生型烟草叶片的质膜相对透性是转基因烟草叶片的1.4倍。     

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.     

Studies on the Chemical Constituents of Tobacco Leaves

S-(?)-2-Isopropyl-5-oxohexanoic acid was isolated from the ether extract of Turkish tobacco leaves. Since 2-isopropyl-5-oxohexanoic acid obtained by ozonolysis of (+)-solanone was leavorotatory, tobacco-derived solanone has R-configuration and might be a precursor of the acid in tobacco leaves.     

The Pool Size of Protochlorophyllide during Different Stages of Greening of Dark Grown Wheat Leaves

The pool size of protochlorophyllide in wheat leaves irradiated for 5 minutes to 6 hours was studied. Protochlorophyllide then accumulated in the dark, but the pool size of regenerated protochlorophyllide was considerably smaller in leaves irradiated for six hours than in leaves irradiated for 5 minutes. The decrease in pool size of regenerated protochlorophyllide was found to take place at the time when the chlorophyll formation had accelerated and reached the linear phase. The protochlorophyllide accumulated is the form with absorption maximum at 650 nm, which is phototransformed to chlorophyllide with maximum absorption at 684 nm. This species goes through the Shibata shift when formed even after 6 hours of irradiation. If leaves, irradiated for 1 or 6 hours, were fed with δ-amino-levulinic acid the protochlorophyllide synthesis was only 1.2 times faster in the leaves irradiated for 6 hours than in those irradiated for 1 hour. In the case of leaves fed with δ-amino-levulinic acid the absorption maximum of protochlorophyllide is at 636 nm and the absorption maximum of the chlorophyllide formed is at 672 nm.     

Catabolic N2-acetylornithine 5-aminotransferase of Klebsiella aerogenes: control of synthesis by induction, catabolite repression, and activation by glutamine synthetase.

Klebsiella aerogenes formed two N2-acetylornithine 5-aminotransferases (ACOAT) which were separable by diethylaminoethyl-cellulose chromatography. One ACOAT was repressed when the cells grew on arginine-containing medium, indicating its function in arginine biosynthesis. The second ACOAT was induced when arginine or ornithine was present in the medium as the sole source of carbon or nitrogen, suggesting its function in the catabolism of these compounds. The induced enzyme was purified almost to homogeneity. Its molecular weight is 59,000; it is a pyridoxal 5-phosphate-dependent enzyme and exhibits activity with N2-acetylornithine (Km = 1.1 mM) as well as with ornithine (Km = 5.4 mM). ACOAT did not catalyze the transamination of putrescine or 4-aminobutyrate. The best amino acceptor was 2-ketoglutarate (Km = 0.7 mM). ACOAT formation was subject to catabolite repression exerted by glucose when ammonia was present in excess. When the cells were deprived of nitrogen, ACOAT escaped from catabolite repression. This activation was mediated by glutamine synthetase as shown by the fact that mutants affected in the regulation or synthesis of glutamine synthetase were also affected in the control of ACOAT formation.     

烟草气孔特性、抗氧化酶活性与臭氧伤害的关系

 2001～2002连续两年在大田生产条件下研究了10个烤烟基因型气孔密度和气孔导度的变异情况及其与抗臭氧伤害的关系;2002年在控制条件下采用低温加臭氧的方法处理烟草植株,研究分析了烟草叶片遭受臭氧伤害后抗氧化酶活性的变化及其与抗臭氧伤害的关系。结果表明：烤烟不同基因型对臭氧伤害的抗性存在显著的差异。烟草叶片下表皮气孔密度和气孔导度与臭氧伤害引起的气候斑点病具有相关关系,在烟株下部叶中,其相关性分别达到显著（R2001＝0.68,R2002＝0.65）和极显著水平（R2001＝0.87,R2002＝0.80）。因此,下部叶气孔密度和气孔导度可以作为抗病育种的选择指标。在控制条件下的试验结果表明：只有低温加臭氧复合因子才能诱发烟草叶片产生臭氧伤害症状。烟草叶片低温下遭遇臭氧伤害后,超氧化物歧化酶（SOD）活性升高,升幅与气候斑点病病情指数负相关,过氧化氢酶（CAT）活性略有升高,过氧化物酶（POD）活性急剧下降。抗性较差的烟草基因型抗氧化酶反应不敏感,臭氧伤害症状严重,抗性较强的烟草基因型抗氧化酶反应敏感,臭氧伤害症状较轻。低温和臭氧同时作用明显影响了活性氧清除系统,致使系统中酶活性比例失调,POD活性急剧下降,可能是烟草叶片产生臭氧伤害的原因之一,SOD和CAT活性升高对消除臭氧伤害具有一定的防御作用。     

Fe,Mn and the grey effect in hydroponically-cultured flue-cured tobacco

A study of the influence of Fe or Mn (supplied as 5, 20, 35, 50 or 65 ppm) on the grey disorder in flue-cured tobacco (Nicotiana tabacum L.) was conducted with mater-cultured plants grown under greenhouse conditions and fertilized with Hoagland's nutrient solution. The solution was highly unbuffered as is the podzolic soil in which grey tobacco plants grow. When plants were treated with 5 to 35 ppm Fe, the trend in grey index (a measure of grey tobacco symptoms) in top and middle leaves was similar to the trend in Fe content of tobacco leaves. The highest grey index was found in these leaf positions with the 35 ppm Fe treatment. When Fe in solution was increased from 35 to 65 ppm grey index in top leaves decreased but was unchanged in middle leaves. Leaf Fe content for these leaf positions changed little. No apparent relationship between grey index and Fe in bottom leaves was observed. Initially, pH of the nutrient solution increased from a mean of 4.75 to 6.50 (indicating preferential anionic absorption) followed by an abrupt decrease in pH to 4.2 (suggesting preferential cationic absorption which included the Fe treatment in solution). The grey effect identical to that found in field-grown plants developed in the cured leaf of plants treated with high levels of Fe and was considered to be one of the major causal agents in grey flue-cured tobacco. None of the levels of Mn induced the grey effect.     

Chemical treatment of soil to prevent transmission of tobacco rattle virus to potatoes by Trichodorus spp

Alternaria longipes (Ell. &Ev.) Mason survived on autoclaved maize stems for 6 months without losing its pathogenicity, but rapidly lost viability on non-autoclaved stems and could not be re-isolated 4 months after inoculation. In laboratory tests it infected both living and dead maize leaves. Some Alternaria isolates from non-solanaceous hosts infected tobacco leaves kept at high humidities for 10 days after inoculation, but not when this incubation period was reduced to 48 h. In the field, perennation on plants other than tobacco is unlikely to be important as a source of inoculum. Pathogenicity of Alternaria isolates was maintained from one season to the next when stored as conidia in sterile soil, or as dried, infected tobacco leaves; some isolates maintained on agar slopes under oil were still pathogenic after 5 years. Alternaria conidia collected from the surface of tobacco seedlings, and isolates from apparently healthy seedling leaves were pathogenic to mature tobacco. In the field conidia were detected on tobacco leaves soon after these emerged, and epiphytic colonies were occasionally found well in advance of symptoms. Many latent infections were also detected up to 5 weeks in advance of symptoms. Visual development of latent infections closely coincided with the end of leaf expansion.     

Inducible and repressible acetylornithine delta-transaminase in Escherichia coli: different proteins

Two acetylornithine δ-transaminases which have different physical and kinetic properties have been isolated from a mutant of E. coli W. Sephadex gel filtration has shown the molecular weight of one transaminase to be approximately 119,000; the second transaminase has a molecular weight of about 61,000. The two transaminases can be separated by ammonium sulfate fractionation. The K_m values of the smaller and larger molecular-weight species for N^α-acetylornithine are 3.1 mm and 1.3 mm, respectively. The K_m for α-ketoglutarate is 1.1 mm for both enzymes. The presence of arginine in the growth medium represses the synthesis of the 119,000 molecular-weight transaminase and induces the synthesis of the 61,000 molecular-weight species.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

湖南烟区土壤交换性钙、镁含量及对烤烟品质的影响

分析了湖南烟区主要土壤类型交换性钙、镁元素含量状况及其对烟叶品质的影响,结果表明:(1)土壤交换性钙、镁含量在不同土壤类型间存在显著性差异,交换性钙含量平均为8.87cmol/kg,以红壤含量最高;交换性镁含量平均为1.16cmol/kg,以黄棕壤含量最高;交换性钙镁比值大小依次为:红壤(11.74)>水稻土(10.25)>黄壤(6.84)>黄棕壤(6.14),在烟叶实际生产中,应重视镁肥在红壤和水稻土中的施用;(2)烟叶钙含量偏高(21.93g/kg±4.37g/kg),烟叶镁含量偏低(2.52g/kg±1.26g/kg),两者均存在广泛的变异性;(3)整体来看,烟叶钙含量随土壤中交换性钙含量的升高和镁含量的降低而显著升高;烟叶镁含量随土壤交换性镁含量的升高而升高,与土壤交换性钙含量的相关性不显著;(4)典型相关分析表明,土壤中交换性镁含量的降低可能引起烟叶钾含量的提高,从而使得烟叶钾素和镁素含量达到较好的平衡;(5)土壤交换性钙、镁含量与烟叶其它化学成分指标的相关分析表明,土壤交换性钙有利于烟株对硼和氯素的吸收,对氮、锌和锰素的吸收则有显著的抑制作用;而土壤交换性镁有利于烟叶总糖、硼素和锰素的积累,对氮、磷、铁和锌素的吸收具有显著的抑制作用。     

烘烤过程中外加淀粉类酶对烤烟淀粉降解的影响

为降低烤后烟叶中淀粉含量,研究了烘烤过程中不同外加淀粉类酶对烤烟淀粉降解的影响。结果表明：烘烤过程中,通过外加淀粉类酶来降解烤烟中的淀粉是有效的。烘烤变黄初期,不同外加淀粉类酶烟叶淀粉降解动态基本一致;变黄中期至定色前期,淀粉降解随外加酶量增加而加剧。烤后烟叶淀粉含量随外加酶量增加而减少,水溶性糖和还原糖含量随外加酶量增加而增加。方差分析表明,不同处理烤后烟叶之问淀粉含量存在极显著差异。多重比较结果表明：K326品种适宜的外加酶量为(6 60)U／g;HD的适宜外加酶量为(8 80)U／g。     

Effect of cyclopentene ��,�¡�-triketone disodium salt on the activity of hydrolases and the state of tobacco mosaic virus particles in tobacco leaves

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.     

PASSAGE OF 5-HYDROXYTRYPTAMINE THROUGH THE BLOOD-BRAIN BARRIER, ITS METABOLISM IN THE BRAIN AND ELIMINATION OF 5-HYDROXYINDO-LEACETIC ACID FROM THE BRAIN TISSUE

Abstract— 5-HT was injected intravenously in rats (10 mg/kg) and a marked increase in brain 5-HT and 5-HIAA was observed. For the first 10 min after injection the penetration of 5-HT into the brain and formation of 5-HIAA is evident. After 10 min degradation of exogenous 5-HT and elimination of 5-HIAA are prominent. Metabolism of exogenous 5-HT in the brain is very fast (half-life between 5 and 10 min; completely metabolized in approximately 80 min). The importance of these results in explaining the permeability of blood-brain barrier to 5-HT is discussed. Experiments on brain slices show that 5-HT is more readily metabolized in brain tissue than eliminated into incubation medium. In contrast, 5-HIAA very easily leaves brain tissue.