F. Pospíšil

Changes in the content of phenolic substances during the growth ofNicotiana tabacum cell suspension culture

The dynamics of changes in the content of four groups of phenolic substances was investigated during the growth cycle of the cell suspension culture ofNicotiana tábacum by means of fractionation. The relative contents of free phenolic acids, their esters, phenolic glycosides, and phenole acids non-extractable with methanol changed in dependence on the growth phase of the culture. A sharp increase, especially in the content of ester- and glycoside-bound phenolics and to a lesser extent also of phenolics belonging to the other two groups, occurred at the end of the lag phase. Then, after a temporary decrease at the early linear phase, the level of phenolics in the three fractions representing bound forms considerably increased again at the late linear and early stationary phases. The synthesized phenolic substances were partially released from the cells into the cultivation medium, which contained 15 to 30 % of the total content of the phenolics in the culture at different phases of the growth cycle. Likely causes of these changes are discussed.

Humic acids with C14

Souhrn1.Listy cukrovky (Beta sacchanfera), jež asimilovaly aktivní kysličník uhličitý C14O2, byly v zemmě podrobeny pochodu humifikace. Po třeeh až pěti měsících humifikace byla usušená zemina extrahována ether alkoholem, pak natrium nebo kalmmhydroxydem a kyselmou chlorovodikovou byly hummové kyselmy oddelěny od fulvokyselin. Všecky frakce byly radioaktivní.2.Rostliny kukuřice (Zea mays) vyrostlé ve zředeném Knopově živném roz toku s přidanou aktivní huminovou kyselmou jevily v kořenech v listech radio aktivitu. Mezi kořeny a listy však byl velký rozdil. Byla-li aktivita kořenů po dvou až dvanácti dnech asi 100 až 200 imp./mm, byla aktivita listů kolem 20 imp./mm počitáno na jednu rostlmu nebo asi 300 imp./min/10 mg u kořenů a na hramci mříitelnosti u listů.3.Když na spodní část rubu prvního listu kukuřice byla nanesena kapka roz toku a suspense aktivní kyselmy hummové, šííla se velmi málo v tomto listu, aktivita druhého a třetího listu byla po pěti dnech na hramci měřitelnosti, obdobně v kořenech. Také z kapilár zabodnutých do mesokotylu se kyselma hummová šííla obdobné, po péti dnech byla na hranici měítelnosti4.Autoradiogramy se plně shodovaly s výsledky získanými pčítačem. Kořeny byly prokresleny zětelne a více meně mtensivně, kdežto listy se neobjevily nebo jen jako slabé stiny.5.Poněvadž aktivita živneho roztoku během kultivace stoupla, nepřijaly rost liny am to množstvi hummové kyselmy, jež bylo rozpuštěno v objemu vytranspiné vody, z toho snad lze soudit, že rostlmy přijímaly velké molekuly, a proto pomalu, Rozhodně nelze rnluvit o hromadění v pletivu.6.Naše pokusy nerozhodují, proniká-li nezměnňná kyselmy huminová do buňky. Můžeme podle mch pouze uzavírat, že, přidáme-li radioaktivní huminovou kyse lmu do vody nebo do živného roztoku, objeví se radioaktivita v koěnech a později slabě l v listech. Připustíme-li že v rostlmných orgánech naměřená aktivita byla působena aphkovanou kyselmou huminovou přímo, znamená to, že promká do rosthny pomalu, není hromaděna a z kořenů se po rostlině šíři pomalu. Je možné, že se radioaktivní uhlík dostane do rostlmy v molekule kyselmy hummové, není však vyloučena možnost, že se do kořenů a z koěnu do listů dostane teprve po rozkladu velké molekuly, eventuálně teprve po její minerahsaci. Ale při rychlé minerahsaci by se v rostlmé jistě šířila rychleji.Summary1.Leaves of sugar-beet (Beta saccharifera), which had assimilated active carbon dioxide C14O2, were subjected to the process of humification in soil. After three to five months of humification the dried soil was extracted with ether-alcohol, then with sodium or potassium hydroxide; the humic acids were separated from fulvic acids with hydrochloric acid. All fractions were radioactive.2.Maize plants (Zea mays) grown in diluted Knops solution with the addition of active humic acid showed radioactivity in the roots and leaves. There was, however, a marked difference between the roots and the leaves. While the activity of the roots after two to twelve days was about 100 to 200 cpm., that of the leaves was about 20 cpm. as calculated for one plant, or about 300 cpm./10 g. for roots and at the limits of measurement for leaves.3.When a drop of solution and suspension of active humic acid was placed on the lower surface of the first leaf, it spread very little even in this leaf; the activity of the second and third leaves was at the limits of measurement after five days; the same applies to the roots.4.Autoradiograms were fully in keeping with the results obtained with the counter. Roots were clearly marked and more or less intensely, while the leaves did not appear or appeared only as faint shadows.5.These experiments do not resolve the question of whether unchanged humic acid penetrates into the cells. It can only be concluded, on the basis of these experiments, that if radioactive humic acid is added to water or to nutrient solution, radioactivity appears in the roots and later weakly in the leaves. If it is assumed that the activity measured in the plant organs was caused by humic acid applied direct, this means that it penetrates into the plant slowly, it is not accumulated and spreads throughout the plant slowly from the roots.Резюмс1.Листья сахарной свеклы (Веta saccharifera), которые асимилировали активный углекислый газ С14О2, пдвергались пропессу гумиϕикании в земле. После 3-5-месячной гумиϕикаци высушенная пчва экстрагировалась смесью эϕира со спиртом и потом гуминовые кислоты отделялись от ϕульвокислот с помощью едкого калия, или едкого натриа или соляной кислоты. Все ϕракции окаэывались радиоактивными.2.У растений кукуруэы (Zea mays), выращенных в раэведенном питательном растворе Кнопа с прибавлением активной гуминовой кислоты. В корнях и листьях наблю-далась радиоактивность. Однако между корнями и листьями была большая разница Если активность корней через 2–12 дней составляла около 100–200 имп./мин., то активность листьев была около 20 имп./мин. В пересчете на 1 растение, или же около 300 имп./мин./10 мг у корней и только следы активности наблюдались у листьев.3.Когда на нижнюю часть обратной стороны первого листа кукурузы наносили каплю раствора и суспензии активной гуминовой кислоты, активность весьма незначи-тельно раснространялась и в этом листе. Актнвность второго и третьего лисета через 5 дней бывала на пороге измераемости так же и в корнях. Из капилляров, воткнутых в мезокотиль, гуминовая кислота раснространяась таким же образом и через 5 дней набльюдались лишь следы активности.4.Аотрадиограммы полноствю соответствовалп результатам, полученным с помощью Корни вырисовывались отнетливо и более или менее интснсивно, тогда как листвя не появлялись только в виде слабых теней.5.Так как активность питательного раствора в тенение культивации повышаласн, растения не впитали даже того количества гуминовой киелоты, которое было растворено н объеме воды, выделившейся при транспирации. Можно иолагать, что в растения поступали крупные молекулые и ноэтому скорость их поступлепия была медленной. Во всяком слунае нельзя говорить о накоплении в тканях.6.Наши опыты не дают ответа, проникает ли гуминовая кислога в клетки в неизме-ненном виде. Можно толвко сказать, что, если радиоактивную гуминовую кислогу прибанить к воде или к питательному раствору, то радиоактивность появляется в корнях и позднее и елабее-и в листьях. Даже если допустить, что отмеченная нами в органах растений активность была обусловлена прямо примененением гуминовой кислоты, это означает, что гуминовая кислота проникает в растсния медленно, не накапливаетея и из корней медлснно распостраняется но всему растению. Возможно, что радиоактивный углерод попадает в растснис в молекуле гуминовой кислоты, но не исключена возможность, что он попадает в корни и из корней в листья только после распада крупной молекулы, или же только после ее минерализации. Одноко нри быстрой минерализацпи активность, несомненно, распространялась бы в растении быстрее.

Screening of compounds for antimutagenic properties towards dimethylnitrosamine-induced mutagenicity inArabidopsis thaliana

The inhibitor of microsomal monooxygenase activities 9-hydroxyellipticine markedly inhibited the frequency of embryonic and chlorophyll mutations induced by the metabolism-requiring mutagen dimethylnitrosamine (DMN) in a higher plantArabidopsis thaliana, but had no effect on the frequency of mutations induced by the direct-acting mutagen N-methyl-N-nitrosourea (MNU). The presence of thiol containing compounds thiourea and cysteine before and during DMN or MNU treatment resulted in a decline of mutation frequency as compared to treatment with either nitroso compound alone. In contrast, the mutagenicity of DMN and. MNU was not altered in the presence of two other thiol compounds, thiouracil and glutathion and by six naturally occurring phenolic acids: ellagic. vanillic, cinnamic, coumaric, caffeic and ferulic acids.

Oxidation of gallic acid by an enzyme preparation isolated from the culture medium ofNicotiana tabacum cell suspension

The oxidation of gallic acid by an enzyme preparation isolated from the culture medium ofNicotiana tabacum cell suspension could be followed by means of fast scan differential pulse polarography (DPP). A product was formed which could be reduced by means of DPP at- 0.47 V (MSE). The nature of the oxidation products is discussed.

The effect of humic acids, fractionated according to molecular mass, on the formation and mutagenicity of N-methyl-N-nitrosourea

Fractions of humic acids (HA-K+) of molecular mass between 500 and 300 000 and exceeding 300 000 showed a very high nitrite depleting ability, whereas the fraction of HA-K+ with molecular mass lower than 500 had little or no such effect. Autoclaving HA-K+ (121 °C, 20 min) decreased the nitrite-depleting ability to about a half. This observation correlates with the results of mutagenic studies in Arabidopsis thaliana, demonstrating the inhibitory effect of HA-K+ and its fractions on the formation of mutagenic N-methyl-N-nitrosourea (MNU) from the nitrosation mixture of N-methylurea and nitrite. Nonfractionated HA-K+ had no inhibitory effect towards the mutagenic activity of preformed direct acting MNU or towards the activation of the promutagen N-nitrosodimethylamine to a mutagenic product.

Anodic oxidation of coumarie and caffeic acids and their effects on nitrate uptake and nitrate reducta se inNicotia na tabacum cell suspension

Anodic oxidation of coumaric acid led to the inhibition of the process at the electrode due to a film which was formed after one-electron oxidation of the acid to phenoxy radical.By contrast, caffeic acid is oxidized in two steps-the phenoxy radical is formed in the first step, quinone in the second step. The inhibition of nitrate uptake by coumaric and caffeic acids is dependent on their concentration. 10-4 M eaffeic acid totally inhibited nitrate uptake and the growth ofNicotiana tabacum cell suspension. 10-6 M caffeic acid markedly inhibited nitrate uptake especially in the first three days after inoculation. 10-6 M coumaric acid did not affect nitrate uptake and nitrate reductase activity, 10-4 M coumaric acid inhibited nitrate uptake by day two after inoculation. Nitrate reductase synthesis correlated with the inhibition of nitrate uptake. Differential effects of coumaric and caffeic acids are explained on the basis of different products of their electrochemical oxidation.

Effect of humic acids on the inhibition of pea choline esterase and choline acyltransferase with malathion

Humic acids in a concentration of up to 100 μg ml-1 affect the activity of choline esterase of pea only weakly during sinapin (choline ester of sinapic acid) hydrolysis. The inhibition is stronger at higher concentrations. A similar course of inhibition with humic acids was also observed during the synthesis of sinapin catalysed with choline acyltransferase present in the same enzyme extract. The organophosphate malathion is a strong inhibitor of both the hydrolysis and the synthesis of sinapin. Thus, for example, at a 3.5 × 10-5 M concentration it causes 85% inhibition of pea choline esterase. However, in combination with 100 μg of humic acids this inhibition decreases to about 8%. It was observed that humic acids prevent the inhibition of both the hydrolysis and the synthesis of sinapin with malathion even at low concentrations. The interaction of the organophosphate malathion with humic acids seems specific. Humic acids do not affect the inhibition of choline esterase with physostigmine or the inhibition of the hydrolysis and the synthesis of sinapin with chlorocholine chloride, alar and hexamine either. This is also true of the activation of the processes with the K+ ions. Humic acids represent competitive inhibitors of choline esterase. Malathion shows an acompetitive — noncompetitive character of the inhibition. The interaction of humic acids with malathion in the roots of the plants may also be assumed in the soil when malathion is applied as an insecticide.

Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.

Influence of physiologically active substances of the soil humus on the activity of glucose-6-phosphate-dehydrogenase in pea (Pisum sativum L.) roots

The influence of phenolic and humic acids on the activity of glucose-6-phosphate-dehydrogenase in the roots of pea under aseptic conditions has been investigated. It seems clear that vanillic and protocatechuic acids inhibit the enzyme activity in the excised roots of pea, but their dry weight increases in relation to the control. Gallic acid stimulates the G-6-PD activity in the roots of whole plants. The humic acids influence neither the enzyme activity nor the dry weight of pea seedlings after short-term treatment.

Changes of 3,4-dihydroxybenzoic and 4-hydroxybenzoic acids inNicotiana tabacum cell suspension culture

The optimum conditions for cell suspension culture experiments have been characterized by respiration,p-Phenylenediamine oxidase and growth of cell suspension ofNicotiana tabacum for 15 days in stationary culture. Change of 3,4-Dihydroxybenzoic and 4-Hydroxybenzoic acids both present at 10−4 M in culture medium were studied in short term experiments. An oxidation product of 3,4-Dihydroxybenzoic acid characterized by an absorption maximum at 280 nm was isolated after 1 h of incubation. The 4-Hydroxybenzoic acid did not change when incubated for 4 h under the same conditions.