Isolation and Identification of Nicotinic Acid as a Flower-Inducing Factor in Lemna

Extracts of flowering plants of the long-day plant Lemna gibbaG3 and the short-day plants Lemna paucicostata 151 and 381 weretested on L. paucicostata 151 for flower-inducing activity.Crude extracts failed to show any activity but after severalpurification steps three fractions with flower-inducing activitywere obtained. One fraction obtained from all three plants wasshown to contain nicotinic acid by mass spectroscopic and NMRspectroscopic analyses. These results raise the possibilitythat nicotinic acid may act to influence the flowering processin Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

Effect of L-Pipecolic Acid on Flowering in Lemna paucicostata and Lemna gibba

L-Pipecolic acid was found to be effective in inducing floweringof Lemna paucicostata 151, 381, 441 and 6746, and of Lemna gibbaG3. When the plants were grown on half-strength Hutner's medium,L-pipecolic acid caused profuse flowering of L. paucicostata151 maintained under 9 and 10 h of light daily. In L. paucicostata441 and 6746, L-pipecolic acid had a strong flower-promotingeffect under a near critical photoperiod. In L. paucicostata381, by contrast, L-pipecolic acid had only a very small effecton flowering. In L. gibba G3 substantial promotion of floweringwas observed under continuous light. When one-twentieth-strengthHutner's medium was used as the basic medium, L-pipecolic acidstimulated flowering in all strains of Lemna examined, evenunder continuous light. When L. paucicostata 151 was grown on one-tenth-strength M mediumor one-twentieth-strength Hutner's medium, the flower-inducingactivity of L-pipecolic acid was greatly enhanced by cytokininunder continuous light. However, when this strain was grownwith 9 h of illumination daily, this synergistic effect of cytokininwas only slight. A short-term (even 1-h) treatment with L-pipecolicacid resulted in flowering, suggesting that L-pipecolic acidis involved in the induction of flowering, rather than its evocation.D-Pipecolic acid also had flower-inducing activity, but itsactivity was 50 times lower than that of the L-isomer. (Received January 23, 1992; Accepted March 9, 1992)     

Flower-Inducing Activity of Benzoic Acid Derivatives for Lemna minor

Lemna minor, strain M601, scarcely flowers in full or 1/10 strengthM medium supplemented with 1% sucrose either in long-day orshort-day conditions, but easily flowers when some benzoic acidderivatives are added to 1/10 strength M-sucrose medium. Thepresence of cytokinin in the medium improves the activity ofbenzoic acid but not significantly that of salicylic acid. Thestructure-activity relationships for the flower-inducing activityof substituted benzoic acid derivatives in the presence of cytokininwere analyzed quantitatively in terms of physicochemical substituentparameters. In the presence of cytokinin, the flower-inducingactivity is determined by electronic and steric effects of thesubstituent. The higher the electron withdrawing ability andthe lower the bulkiness of the substituent, the higher is theactivity. In the absence of cytokinin, the activity is enhancedby higher electron withdrawing ability of the substituent, butthe steric effect is insignificant. In both conditions, thehydroxyl group in the ortho position exhibits an additionaleffect to enhance activity, and the molecular form responsiblefor the activity seems to be undissociated neutral species.The structure-activity relationship in L. minor M601 is basicallysimilar to that for L. paucicostata 151. (Received February 2, 1983; Accepted May 2, 1983)     

Conversion of Lysine to L-Pipecolic Acid Induces Flowering in Lemna paucicostata 151

The natural occurrence of L-pipecolic acid and conversion oflysine to L-pipecolic acid in Lemna paucicostata 151 were demonstrateddefinitively by GC-MS. The strong flower-inducing activity ofL-pipecolic acid has already been demonstrated. Thus, the presentstudy indicates that the effect of lysine on flowering is mediatedby L-pipecolic acid. (Received June 30, 1997; Accepted August 22, 1997)     

Flower-Inducing Activity of Water Extract of Lemna

Flowering of Lemna paucicostata 441 (P441), a sensitive short-dayplant (SDP), was promoted under a near critical photoperiodby the crude water extract of the same plant added to the medium.The extract induced flowering in L. paucicostata 151 (P151),a weakly responsive SDP, under continuous light. The activityfor P151 was greatly promoted by simultaneous application ofbenzyladenine, and the extract of only 0.3 mg fr wt plant addedto 10 ml of assay medium with 1 µM benzyladenine was active.Active substance(s) was similarly obtained from both flower-inducedand non-induced plants, and more or less from all species andstrains of Lemna tested, including P151. However, the extractof short-day strains was more active than that of L. gibba G3(G3), a long-day strain. G3 responded only slightly to the extractof either P441 or G3, whereas P151 responded far more stronglyto the extract of P441 than to that of G3. (Received April 17, 1989; Accepted August 10, 1989)     

Flower-Inducing Activity of Lysine in Lemna paucicostata 6746

Flowering of Lemna paucicostata 6746, a typical short-day plant,was induced by culture for 96 or 120 h in nitrogen-free mediumunder continuous illumination. To examine the effects of lysine,we homogenized entire plants of L. paucicostata 151 in a solutionof lysine and the supernatant obtained after centrifugationof the homogenate was added to the medium to give various concentrationsof lysine in the medium. Flowering of strain 6746 in nitrogen-freeor nitrogen-deficient culture medium was effectively promotedby the addition of a lysine-containing supernatant to the medium.The suppressive effect of elastatinal, a protease inhibitor,on the induction of flowering was almost completely reversedby the simultaneous application of a lysine-containing supernatantto the medium. During nitrogen-free culture, the level of endogenousfree lysine, expressed on the basis of the amount of total freeamino acids, increased. Lysine-containing supernatants alsoinduced flowering of plants in nitrogen-rich medium under continuousillumination. These findings suggest that endogenous lysineis involved in the induction of flowering in L. paucicostata6746 on nitrogen-free or nitrogen-deficient medium, as it isin the induction of flowering in L. paucicostata 151 (Received July 29, 1996; Accepted November 18, 1996)     

Interaction between L-Pipecolic Acid and Water Extracts of Various Plant Species in Floral Induction of Lemna paucicostata

The flower-inducing activity of L-pipecolic acid was synergisticallyenhanced by simultaneous application of the water extracts ofLemna paucicostata and Pharbitis nil, but suppressed by thewater extracts of all other plants we examined. Simultaneousapplication of the water extract of Lemna enhanced the flower-inducingactivity of all plant water extracts. (Received June 6, 1990; Accepted July 7, 1990)     

Production of the Water-Extractable Flower-Inducing Substance(s) in Lemna

Crude water extract of Lemna paucicostata Hegelm., strain 441,had high flower-inducing activity. This activity was heat-stable,but water extract of this plant after heat treatment had verylow activity. The water extract heated immediately after homogenizationalso had low activity, and this activity increased rapidly duringincubation of the plant homogenate before heating even at 0?C.The increase in activity did not occur during separate incubationof the supernatant and pellet obtained by centrifugation ofthe homogenate; some reaction between the components of thesupernatant and pellet may be necessary for production of theheat-stable flower-inducing substance(s). Oxygen deprivation(incubation in nitrogen gas) or presence of ascorbate duringincubation of the plant homogenate markedly lowered the generationof the flower-inducing activity. These results suggest thatthe active substance(s) is produced by an oxidative reaction.No significant difference could be found between photoperiodicallyinduced and non-induced plants in the activity of the waterextract after heat treatment. (Received April 9, 1990; Accepted July 5, 1990)     

Isolation and Identification of Lutein from Lemna paucicostata 381 as an Inhibitor of Benzoic Acid-induced Flowering in Lemna paucicostata 151

Efforts were made to isolate flower-inhibitory substances from extracts of the short-day plant Lemna paucicostata 381. Lemna paucicostata 151, which was used in the bioassay, exhibits poor flowering in response to the photoperiod, but flowers profusely in response to benzoic acid. Therefore, only those substances that inhibit benzoic acid-induced flowering were studied. Several fractions obtained by silica gel column chromatography exhibited flower-inhibitory activity when tested on L. paucicostata 151. After several purification steps, one of the active principles was identified as lutein by MS, UV and NMR spectroscopic analyses. Lutein and its isomer zeaxanthin inhibited benzoic acid-induced flowering in both L. paucicostata 151 and 381.     

Flower-Inducing Activity of Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Deficient Medium

Lemna paucicostata 151, a weakly responsive short-day plant,flowers even under continuous illumination when cultured onnitrogen-free medium for more than 72 hours with subsequentculture on nitrogen-rich medium. During the nitrogen-free culture,the protease activity and protein content of the plant increasedand decreased, respectively. The plant contained a protein(s)that induced flowering of the plant when added to the medium.The level of this protein(s) also decreased during the nitrogen-freeculture. The total amount of free amino acids in plants culturedon nitrogen-free medium for 96 hours decreased to about 15%of that in plants at the start of nitrogen-free culture, butlevels of some amino acids increased. These amino acids wereexamined for their effects on flowering of plants cultured onnitrogen-deficient or nitrogen-free medium. Most of the aminoacids had no effect on flowering. However, profuse floweringwas induced when lysine was added to the medium. Lysine promotedthe flower-inductive process(es) rather than the developmentof flower buds. These results suggest that nitrogen deficiency-induced floweringof the plants is induced by lysine, which is generated froma specific protein(s) by proteolysis. (Received May 11, 1992; Accepted July 30, 1992)     

Flower-Inducing Activity of Exogenous Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Rich Medium

Flower-inducing activity of lysine was examined in Lemna paucicostata151, a weakly responsive short-day plant, cultured on nitrogen-richmedium under long-day conditions (continuous light). Lemna paucicostata151 was homogenized in a solution of lysine and the homogenatewas centrifuged. The supernatant (lysine-containing extract)was added to nitrogen-rich medium after passage through a membranefilter to give various concentrations of lysine in the medium.Flowering was induced in plants grown for six days on mediumthat contained lysine at concentrations above 0.25 µM.In plants grown on medium that contained 1 µM lysine,a significant flowering response was observed on the fourthday of culture. However, the flower-inducing activity of lysinedisappeared when the lysine-containing extract was added tothe medium and the medium was then autoclaved, suggesting thatthe active principle is unstable to autoclaving. Among derivativesof lysine tested, lysine hydroxamate had the highest flower-inducingactivity and lysyl lysine had almost same activity as that oflysine. When added to the medium without homogenization withplant material, lysine and lysyl lysine had flower-inducingactivity but lysine hydroxamate did not induce flowering. (Received April 26, 1993; Accepted November 8, 1993)     

Increase in the Activities of Protein Kinases under a Flower-Inducing Condition in Lemna paucicostata

The activities of protein kinases and the phosphorylation ofsubstrate proteins were assayed in Lemna paucicostata 6746 culturedunder flower-inducing and non-inducing conditions. The activitiesof two protein kinases in the soluble fraction, one cAMP-activatedand the other cytokinin-inhibited, increased drastically underthe flower-inducing condition. However, no significant differencewas observed in the activities of another protein kinase inthe soluble fraction and a microsomal protein kinase, underflower-inducing and non-inducing conditions. The in vitro phosphorylation of cellular proteins was much greaterin the soluble fraction obtained from plants grown under theflower-inducing condition. Four polypeptides of mol wt 59,000,51,000, 16,000 and 14,000 were highly phosphorylated in thesample from flower-induced plants. No difference in cAMP levelwas observed between flower-inducing and non-inducing conditions.Cyclic AMP (Received April 27, 1987; Accepted October 19, 1987)     

Involvement of Norepinephrine in the Production of a Flower-Inducing Substance in the Water Extract of Lemna

A flower-inducing substance (FIS) is produced by incubationof the pellet of centrifuged ho-mogenates of Lemna paucicostata441 (P441) with commercial enzyme preparations such as catalase,cellulase, lipase and proteinase K. The active component inthese preparations was identified as tyrosine. The tyrosinemetabolites, dopa, dopamine, norepinephrine (NE) and epinephrine,were also effective for the production of FIS, and NE the mosteffective. Addition of only 1 µg NE to the pellet obtainedfrom 100 mg fresh weight of P441 produced FIS without incubation.NE added to the pellet heated after resuspension also producedFIS but that added to the pellet resuspended in boiling waterimmediately after separation did not. A heat-stable substance(s)produced by a heat-unstable reaction in the pellet may reactwith NE to produce FIS or interact with NE to induce flowering.About 400 ng per g fresh weight of NE was detected in the waterextract of P441 plants, but only about 0.5 ng in the hot-acidicwater extract of the plants, which suggests that most of theNE was produced after homogenization. (Received October 17, 1990; Accepted December 28, 1990)     

几株产活性物质的海洋细菌的分离与初步鉴定

从海泥、海水及海洋动物中分离到数十株细菌 ,对其产生活性物质的菌株进一步筛选 ,得到产蛋白酶、淀粉酶和抑菌活性等生物活性物质的几株海洋细菌 ,将其中的活性高的四株菌纯化后进行菌种鉴定。通过对革兰氏染色、个体及群体形态观察、糖类发酵、硝酸盐还原等生理特性的研究 ,依据《伯杰氏细菌鉴定手册》确定它们的分类地位 ,它们分别应归属为欧文氏菌属 (Erwiniasp .)、气单孢菌属 (Aeromonassp .)、微球菌属 ( (Micrococoussp .)、和假单孢菌属 ( (Pseudomonassp .)。     

筛选鉴定一株产生抑菌活性物质的海洋放线菌

目的：分离筛选能够产生抑菌活性物质的海洋放线茵,并进行生理生化和16SrDNA鉴定。方法：用分离培养基培养海洋放线菌,并筛选出能够产生抑菌活性物质的菌株,对所筛选菌株的形态特征、生理生化特性进行鉴定分析;采用通用引物27F、1492R扩增该菌株的16SrDNA,对测序结果进行分析;采用Neighbor—Joining（N—J）法构建系统发育进化树。结果：筛选到一株对金黄色葡萄球菌、大肠杆菌、白色念珠菌具有较强抗性的海洋放线菌F1,该菌株好氧,中度嗜盐,在高氏I号培养基上呈白色绒粉状,16SrDNA序列比对表明该菌株与田无链霉菌（Streptomyces tanashiensis）NR043369的相似度为99％。结论：筛选到的菌株F1是一株海洋来源的放线菌,与田无链霉菌NR043369的同源性较高,可能属海洋链霉菌属,对金黄色葡萄球菌等病原菌具有较强的抑菌活性。     

Isolation and Identification of ( + )-Hexylitaconic Acid as a Plant Growth Regulator

( + )-Hexylitaconic acid was isolated as a root-growth stimulating substance from a culture filtrate of Aspergillus niger K-88.     

Production of Flower-Inducing Substance(s) by the Treatment of Lemna Extract with Some Commercial Enzyme Preparations

Flower-inducing substance(s) (assayed with Lemna paucicostata151) was produced by incubation of the centrifuged pellet ofthe homogenate of L. paucicostata 441 (P441) with the commercialenzyme preparations, catalase, cellulase, invertase, lipase,pectinase, peroxidase and proteinase K. These enzyme preparationswere effective even after autoclaving. The flower-inducing activity of the centrifuged supernatantwas also enhanced by incubation with the cellulase preparation,but that of the heat-treated supernatant or pellet was not.Oxygen deprivation (incubation in nitrogen gas) or presenceof ascorbate during the incubation markedly lowered the generationof the flower-inducing activity. These results suggest thatthe flower-in-inducing substance(s) is produced by an oxidativereaction from some heat-stable component in the commercial enzymepreparations by the action of some heat-unstable factor (enzyme?)in the plant. (Received June 6, 1990; Accepted August 16, 1990)     

产酸性脲酶菌株的筛选、鉴定及其脲酶的应用初探

利用传统微生物筛选方法,从动物粪便中经过酸性平板初筛、中性平板复筛,得到一株产酸性脲酶的菌株JN_R12。通过比较形态特征、生理生化以及16S rDNA测序结果,结合系统发育分析,确定该菌为肠出血性大肠埃希氏菌Enterohemorrhagic Escherichia coli O157：H7。JN_R12所产脲酶对酒精有较好的耐受性。离心后得到的全细胞在模拟酒样中的尿素去除率接近100%;黄酒中24h孵化后的去除率在60%以上。