M. Kamínek

Cytokinin activities ofN6-benzyladenosine derivatives hydroxylated on the side-chain phenyl ring

Cytokinin activities ofN6-benzyladenosine (bzl6Ado) and its derivatives hydroxylated on the side chain phenyl ring inortho, meta, andpara positions were compared in four bioassays based on stimulation of growth of tobacco callus, retention of chlorophyll in excised wheat leaves, dark induction of betacyanin synthesis inAmaranthus cotyledons, and release of lateral buds of pea from apical dominance. In all these bioassays hydroxylation of the phenyl ring of bzl6Ado inortho andpara positions significantly decreased cytokinin activity. Compared with bzl6Ado, the activity was decreased about 10× in the tobacco callus bioassay and wheat leaf chlorophyll retention test, 100× in theAmaranthus betacyanin bioassay, and 20× and 200×, respectively, in the pea bud test. Hydroxylation of the phenyl ring inmeta position increased activity in the tobacco callus, and wheat leaf chlorophyll retention bioassays, 9× and 1.7×, respectively, decreased activity about 2.4× in the pea bud test and was without effect in theAmaranthus bioassay. Cytokinin activity of themeta hydroxy derivative,N6-(m-hydroxybenzyl) adenosine, was as high as that oftrans-zeatin in all four bioassays. Possible regulation of biological activities of cytokinins by positionally specific hydroxylation of the side chain phenyl ring is discussed with respect to the reported occurrence of natural purinyl cytokinins with aromatic side chains.

Quantitative determination of starch, amylose, and amylopectin in plant tissues using glass fiber paper

Methods for accurate and rapid determination of starch, amylose, and amylopectin in plant tissues are described. They are based on simplified extraction of starch with 32% perchloric acid and selective retention of the starch-iodine complex on a glass fiber disk (Whatman GF/A). The starch on the disk is dissolved in 0.75 M sulfuric acid and estimated with phenol. For amylose and amylopectin determination the starch on the disk is dissolved in perchloric acid, precipitated with ethanol, and retained on a 10-cm glass fiber strip. Both polysaccharides are separated by a chromatographic procedure involving development of the strip in a mixture of ethanol and dimethyl sulfoxide and in dimethyl sulfoxide. The strip is washed in ethanol and stained with iodine or used for polysaccharide quantitation. As little as 5 micrograms of starch or its components present in different amounts of plant material can be estimated.

Effect of stereospecific hydroxylation of N6-(Δ 2-Isopentenyl)adenosine on cytokinin activity

The cytokinin activities of cis and trans ribosylzeatin isomers and that of N6-(Δ2-isopentenyl)adenosine were compared in four bioassays. The trans isomer was found to be more active than the cis isomer in stimulation of cucumber cotyledon expansion (100x), retention of chlorophyll in detached leaf pieces (7x), induction and stimulation of chlorophyll synthesis in cucumber cotyledons (20x) and of betacyanin synthesis in Amaranthus caudatus seedlings grown in the dark (60x). The N6-(Δ2-isopentenyl)adenosine adenosine was less active than the trans ribosylzeatin in all four bioassays and more active than the cis ribosylzeatin in induction and stimulation of betacyanin and chlorophyll synthesis. These results show that the hydroxylation of the trans methyl group in the N6 side chain of N6-(Δ2-isopentenyl)adenosine increases the biological activity and that this activity is either decreased or not significantly changed when the cis methyl group is hydroxylated.

Root Formation in Pea Stem Sections and its Inhibition by Kinetin, Ethionine and Chloramphenicol

Root formation in the etiolated pea stem sections and inhibition of this process is described in the present paper. Sodium fluoride, iodoacetic acid, norvaline, phenylserine, 5-bromuracil and 2-thiouracil did not inhibit the root formation completely. Complete inhibition, however, was observed after treatment of pea stem sections by kinetin, ethionine and chloramphenicol (5 X 10-5 M, 1 x 10-2 M, and 1 x 10-2 16 hours after sectioning). The concentration of kinetin which produced complete inhibition of root formation simultaneously stimulated the growth of the lateral buds.Root formation under the conditions described below can be divided into two stages. The first stage 64 hours from the beginning of the experiment, the second stage 64 hours later. Further, the first stage includes the formation of the meristematic cells in the pericycle areas. In the second stage are included the growth of roots and differentiation of root-tissues. Roots were formed, first of all, in the short vertical region of the sections near to the basal buds. Secondary xylem formation was also observed during the cultivation of the sections. This process was stimulated by kinetin.AbstractByla sledována tvorba kofenů v etiolovanych úsecich hrachovych stonků a jeji inhibice. Fluorid sodny, jodoctová kyselina, norvalin, fenyl-serin, 5-bromuracil a 2-tiouracil neinhibovaly úplnĚ tvorbu kořenů. Uplná inhibice tvorby korenú byla pozorována po aplikaci kinetinu, etioninu a chloramfenikolu (5 x 10-5 M, 1 x 10-2 M, 1 x 10-2 M 16 hod. po odřezánÍ úseků). Koncentrace kinetinu působici üplnou inhibici tvorby kořenú stimulovala růst postrannÍch pupenů.Tvorbu kořenů za uvedenÝch podminek lze rozdĚlit na dvĚ stadia. Prvni - 64 hod. od začátku pokusu, druhé - 64 hod. pozdĚji. V prvnÍm stadiu se tvoři meristematické buńky v pericyklu. Ve druhém stadiu rostou kořeny a probÍhá differenciace kořenového pletiva. Nejprve se tvori koreny v krátkém vertikálnÍm pásmu useku poblÍž basálniho pupene. Při pĚstováni úsekо byla rovnĚž pozorována tvorba druhotného xylému. Tento proces byl stimulován kinetinem.AbstractВработе исследовало сь образование корне йи еуо инуибирование вэтиолированных отрезках стебля уоро ха. Фтористый натрий,й одуксусная кислота, норвалин, фенил-серин, 5-бромурацил и 2-тиоурацил не инуибир овали полностью обр азование корней. Полное подавление корнеобразования при применении кин отина, этионина и хлор-амфеникола (5 х 10-5 м, 1 х 10-2 м, 1 х 10-2 м, 16 часов уюсль взя тия отрезков). Концентрация кинет ина, полностью подав ляющая корнеобразов ание, стимулировала рост боковых почек. Корнеобразование в данных условиях мож но разделить на две с тадии. Первая -64

Dynamics of production oftrans-zeatin andtrans-zeatin riboside by immobilized cytokinin-autonomous and cytokinin-dependent tobacco cells

Two strains of cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) differing in their requirement for exogenous cytokinins (cytokinin-dependent and cytokinin-autonomous) were immobilized on polyphenylenoxide (Sorfix) activated with glutaraldehyde. Columns packed with immobilized cells were continually eluted with diluted Murashige and Skoogs medium lacking or supplemented with synthetic cytokinin (6-benzylaminopurine; BA). Purified samples of column eluates were fractionated by HPLC, andtrans-zeatin (t-Z) andtrans-zeatin riboside (t-ZR) content was estimated by enzyme immunoassay. Both cytokinin-autonomous and cytokinin-dependent tobacco cells produced and excretedt-Z and its riboside, and there were significant quantitative differences between the strains. The steady-state excretion rate oft-Z was 19.8 ng · g−1 dw · h−1 and 4 ng · g−1 dw · h−1, respectively, and that oft-ZR 4 ng · g−1 dw · h−1 and 1 ng · g−1 dw · h−1, respectively. Exposure of cytokinin-dependent cells to BA after 72 h of starving for this synthetic cytokinin caused temporary increase in excretion of both zeatin and its riboside. After the application of 5 μM BA for 24 h, the excretion rate oft-ZR reached 5 ng · g−1 dw · h−1 (5-fold increase), and that oft-Z achieved 12 ng · g−1 dw · h−1 (3-fold increase). The elevation oft-Z excretion was delayed about 13 h compared witht-ZR excretion, which started increasing almost immediately after BA application. A pulse of BA in lower concentration (1.5 μM for 30 h) provoked lower response.

Sensitivity of oat leaf chlorophyll retention bioassay to natural and synthetic cytokinins

The cytokinin bioassay based on retention of chlorophyll in excised oat leaf pieces is more sensitive to the synthetic cytokinins (6-benzylaminopurine and kinetin) than to the natural cytokinins (trans-zeatin and N6-[Δ2isopentenyl]adenine). This difference in sensitivity decreases with increasing length of leaf pieces (from 2 to 10 cm) and with increasing volume of application (from 5 μ1 to 25 μl). Application of the cytokinin solution to the basal and apical part of the 8 cm pieces decreases the sensitivity of the bioassay but has no significant effect on the relative activities of trans-zeatin and 6-benzylaminopurine. Using 7 cm pieces and 20 μl of solution the trans-zeatin and N6-(Δ2-isopentenyl)-adenine can be detected at concentrations of 10-5 M and 5 x 10-5 M, respectively.

Reduced chlorophyll synthesis in cytokinin-autonomous strains of tobacco callus

Summary Chlorophyll formation in one cytokinin-dependent (D) and three cytokinin-autonomous (A1, A2 and A3) strains of callus tissue derived from stem pith of tobacco (Nicotiana tabacum L. cv. Wisconsin No. 38) was compared. The tissues were grown 14 days in darkness and then transfered to continuous light. Total chlorophyll content in all A strains (A1, A2 and A3) was significantly lower (14.0 %, 24.7 % and 36.2 %, respectively) than that in the D strain (100%). The number of plastids per cell in A1, A2 and A3 strains (157, 174 and 148, respectively) was reduced as compared with the D strain (210) to a lesser extent. Reduced chlorophyll synthesis in the A strain therefore seems to be mainly responsible for the significant difference in the chlorophyll content rather than plastid number. An attempt was made to increase the chlorophyll formation in the chlorophyll-deficient A1 strain by the addition of kinetin and auxins. Naphthaleneacetic acid stimulated chlorophyll formation in both, A1 and D strain, its optimum concentration (5 × 10−1μM) is much lower than the optimum for maximum tissue fresh weight (5 μM). On the other hand chlorophyll synthesis in both, D and A1 strain, is stimulated by higher concentrations of kinetin (10 μM) than are optima for their growth (1 μM and 10−2 μM, respectively). Addition of auxin or kinetin to medium did not increase chlorophyll synthesis in the A1 strain to the level found in the D strain. The relationship between cytokinin-autonomy and reduction in chlorophyll synthesis is discussed.

Induction of cytokinin-autonomy and chlorophyll-deficiency in tobacco callus tissue by streptomycin

Summary Chlorophyll content in cytokinin-dependent callus tissue ( Nicotiana tabacum L. cv. Wisconsin No. 38) cultivated on medium containing naphthaleneacetic acid (5 μM) and kinetin (2 × 10 −1 μM) was reduced by 85% in the presence of streptomycin (0.1 g/1). Tissue sub-cultured seven times during a nine month growth period on medium containing streptomycin when transfered to medium containing no added streptomycin or kinetin was found to be cytokinin-autonomous but deficient in chlorophyll. Both effects were maintained during successive passages on streptomycin- and kinetin-free medium for the past year. The chlorophyll content in the new streptomycin-induced cytokinin-autonomous strain (A S strain) was permanently decreased to one third the control value and the number of plastids in A S strain was lower than that found in the cytokinin-dependent strain (121 and 210 plastids per cell, respectively). Simultaneous induction of cytokinin-autonomy and reduction of chlorophyll formation by streptomycin may indicate that synthesis of chlorophyll in functioning plastids significantly affects the cytokinin-economy of the cell.

Acropetal transport of kinetin in pea stem sections

Pomocí stimulace růstu postranních pupenů etiolovaných řízků hrachu kinetinem bylo zjištěno, že biologický účinek kinetinu není omezen pouze na místo jeho aplikace, ale že se může projevit i v jiných částech řízků. Tento jev si autor vysvětluje transportem kinetinu.AbstractПри помощи стимулирования роста боковых почек этиолированных осевых секций гороха кинетином установлено, что биологическое действие кинетина не ограничено лишь местом его применения, но может проявиться и в других частях секций. Это явление автор объясняет транспортом кинетина.

Catabolism of glucose in pea stem section during root formation and its inhibition by kinetin and ethionine

The catabolism of specifically14C-labelled glucose during the root formation and its inhibition by kinetin and ethionine in the etiolated pea stem sections were studied. The formation of root meristematic foci in the pericycle region of sections was accompanied by the decrease of the C6/C1 ratio. Such a result and activation of pentose phosphate cycle, which was also checked by another method, suggested increased participation of pentose phosphate cycle in glucose oxidation. The above mentioned changes were also found after the prevention of root formation by ethionine and, therefore, do not seem to be specific for the meristematic foci formation. The growth of newly formed roots was closely connected with the rise of C6/C1 values.The increase of CO2 release from the first carbon atom of glucose molecule was recorded after the inhibition of root formation by ethionine.The rise of C6/C1 values and decrease of pentose phosphate cycle activity was observed after the treatment of pea stem sections by kinetin in the first 64 hours after sectioning. In this case root formation was prevented and the growth of lateral buds was stimulated. The secondary xylem formation which took place later was accompanied by the activation of the pentose phosphate cycle. These phenomena are discussed in relation to cell division and biosynthesis of lignin-precursors.AbstractByl studován katabolismus specifioky14C značené glukosy při tvorbě kořenů a její inhibici kinetinem a ethioninem v ethiolovaných stonkových ůsecích hraohu. Tvorba meristematických základů korenů v pericyklu ŭseků byla doprovázena snížením poměru C6/C1. Tento výsledek a aktivace pentosofosfátového cyklu, zjiřtěná také jinou metodou, svědčí o zvý sené ŭčasti pentosofosfátového cyklu v oxidaci glukosy. Uvedené změny byly zjiřtěny také půi zabránění tvorby korenů ethioninem a tudíž nejsou specifické pro tvorbu meristematických základů. BŮst nové utvářených kořenů byl vždy spojen se zvýšením hodnoty C6/C1.Zvýření vzniku CO2 z prvního uhlíkového atomu glukosy bylo pozorováno po inhibici tvorby korenů vlivem. ethioninu. Zvětšení hodnoty C6/C1 a snížení aktivity pentosofosfá tového cyklu bylo pozorováno po aplikaci kinetinu na ŭseky hrachových stonků během prvních 64 hodin po jejich odřezání. V tomto případě byla tvorba kořenů inhibována a růst postranní ch pupenů byl stimulován. Pozdější tvorba druhotného xylemu byla doprovázena aktivací pentosofosfátového cyklu. Uvedené jevy jsou diskutovány ve vztahu délení buněk biosynthese prekursorů ligninu.AbstractИзучался катаболизм специфиески C14 меченн ой глюкозы при образо вании корней и при его ингибирован ии кинетином и зтиони ном в этиолированных отрезках стебля гороха. Образование м еристематических ос нований корней в пери цикле отрезков сопровождалось пони жением отношшения C6:C1. Этот результат и акти вация пентозо-фосфатного цикла уст ановленная также и др угим методом свидете лъствует также о повыпШепном участи и пентозофосфатного цикла в окислении глю козы. Приведенные изменения были устан овлены также при инги бировании образован ия корней зтионпном и таким образом не явл яются специфическим и для образования мер истематических образований. Рост нов ообразующихся корне й был во всех случаях с вязап с повьпне-нием отношшения C6:C1.Повьпшенное образов ание СCO2 из нервого ато ма углерода наблюдал ось после инги-бирования образован ия корней зтионином. П овышение отношШения C6:C1 и понижение активности пентозоф осфатмого цикла прои сходило при применен ии кинетина к отрезка м гороховых стеблей в п ериод 64 часов после их отрезки. Б зтом случае образование корней ингибировало съ и рост боковых поче к стимулировался. Бол ее позднее обра-аование вторпчной кс илемы сопровождалос ь активированием пен тозофосфатного цикл а. Приведенные явления обсуждаются по отно.ш еяию к делению клеток и к биосинтезу предшественников ли гнина.