Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Increase of cytokinin activity in detached etiolated cotyledons of squash after illumination

Cytokinins nullified the lag period of the formation of chlorophyllin detached etiolated cotyledons of squash (Cucurbita moschataDuch. var. melonaeformis Makino cv. Tokyo). One hour after illumination,cytokinin activity in detached cotyledons rapidly increasedand maintained a certain level for another hour. ¹ Present address: Department of Enviromental Studies, Collegeof Integrated Arts and Sciences, Hiroshima University, Hiroshima730, Japan. (Received November 25, 1976; )     

The relationship between transpiration and chlorophyll synthesis in etiolated squash cotyledons

Etiolated cotyledons of squash (Cucurbita moschata Duch. var.melonaeformis Makino cv. Tokyo) treated with kinetin, KCl orfusicoccin in the dark increased both transpiration in the darkand chlorophyll synthesis after exposure to light. The minimumperiod of kinetin treatment to stimulate transpiration was similarto that to accelerate chlorophyll synthesis. On the other hand,treatment of cotyledons with vaseline or abscisic acid markedlyinhibited both transpiration and chlorophyll synthesis, suggestingthat cytokinin-induced stimulation of chlorophyll synthesiscould be mediated through the effect on the stomatal opening. (Received December 7, 1977; )     

Formation of Chlorophyll-Protein Complexes in Greening Cucumber Cotyledons in Light and then in Darkness

The changes in chlorophyll-protein complexes (CPs) in cucumbercotyledons during illumination and subsequent dark incubationwere studied by SDS-polyacrylamide gel electrophoresis. Whenetiolated cucumber seedlings were illuminated, chlorophyll wassynthesized and CPs were formed. In the early phase of greening(6 h of illumination), light-harvesting chlorophyll a/b-proteincomplex (LHCP) was the main GP. As the greening proceeded, P700chlorophyll a-protein complex (CP1) accumulated. When 6-h illuminatedseedlings were transferred to darkness, CP1 accumulated concomitantlywith a decrease in LHCP without new chlorophyll synthesis. Thechanges in the amounts of CPs in the dark became smaller withthe progress of greening and were not observed after 72 h ofillumination. These changes were confirmed by examining thechlorophyll/P700 ratio and the low temperature absorption spectrumof cotyledons. These results suggest that in the early phaseof greening, CPs were unstable and their chlorophyll moleculeseasily exchanged with those of other kinds of CPs. (Received October 14, 1982; Accepted December 1, 1982)     

Effects of Compounds that Influence Calcium Absorption and of Calcium-Calmodulin Inhibitors on Zeatin Induced Growth and Chlorophyll Synthesis in Excised Cucumber Cotyledons

Previous results indicate that cytokinin action requires formationof Ca-calmodulin. We evaluated that hypothesis for cytokinin(zeatin) induced growth and chlorophyll formation in excisedcucumber (Cucumis sativus L.) cotyledons using several compoundsexpected to increase or decrease cytosolic levels of Ca²⁺ andthereby perturb formation of Ca-calmodulin. We also studiedeffects of eight drugs known to inhibit action of Ca-calmodulinin animals. Most of the results indicate no special requirementof Ca-calmodulin for cytokinin action in these organs. (Received November 24, 1988; Accepted April 24, 1989)     

Low Temperature Treatments Induce an Increase in the Relative Content of Both Linolenic and {lambda}3-Hexadecenoic Acids in Thylakoid Membrane Phosphatidylglycerol of Squash Cotyledons

The effect of low temperatures on the fatty acid compositionof phosphatidylglycerol (PG) in thylakoid membranes, in particularon the ratios of nmol% 16:1(3t) (mg fresh weight)^–1 ofcotyledons and nmol 16:1(3t) (mg chlo rophyll)^–1 weremeasured during squash seedling growth. Plants were germinatedand grown for one day at 30°C then were either kept at 30°C(control plants) or trans ferred to low temperatures (18, 14or 10°C). When plant were transferred from 30°C to lowtemperatures, the increase in fresh weight was gradually limited.The lowe the temperature, the smaller was the fresh weight.In contrast, the relative content of 16:1(3t) and 18:3, as wella the ratios of nmol 16:1(3t) (mg chlorophyll)^–1 and mol%16:1(3t) (mg cotyledon fresh weight)^–1 increased indicatingthat the increase of fresh weight and chlorophyll was mor sensitiveto low temperature than PG desaturation in thyla-koid membranes.Furthermore, low temperatures inducei an increase in 16:1(3t)and 18:3 (the final products of PC synthesis) at the expenseof 16:0 and 18:1 (the initial products of PG synthesis). However,within a range of temperature from 10 to 18°C, the extentof these changes (nmol% of 18:3 or 16:1(3t) per day) was graduallylimited by lower temperatures. We therefore propose that lowtemperature inhibit both fatty acid synthesis and desaturationactivities. However, at low temperatures the fatty acid synthesisis likely to be more strongly inhibited than the desaturationactivities, thus explaining the observed increase in the relativecontent of PG-18:3 and PG-16:l(3t). Results an discussed interms of the mechanism which could be in volved in the metabolismof PG in squash cotyledons. (Received July 5, 1996; Accepted March 10, 1997)     

Lepidimoide Promotes Light-Induced Chlorophyll Accumulation in Cotyledons of Sunflower Seedlings

The effect of disaccharide lepidimoide on light-induced chlorophyll accumulation was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings and detached cucumber (Cucumis sativus L.) cotyledons. From studies on the structure-activity relationships of lepidimoide, its analogs, and sucrose with respect to light-induced chlorophyll accumulation in the cotyledons of sunflower seedlings, both lepidimoide and the free carboxylic acid of lepidimoide (lepidimoic acid) showed the highest promoting activity, whereas the hydrogenated lepidimoide, which lacks a double bond in the C4, 5 position in uronic acid, showed lower activity than lepidimoide; however, sucrose exhibited very weak activity. These results suggest that lepidimoide acts as a new type of plant growth regulator, not simply as a carbon source providing energy. Lepidimoide promoted not only light-induced chlorophyll accumulation in sunflower cotyledons but also light-induced 5-aminolevulinic acid content, which is considered to be a rate-limiting step in chlorophyll biosynthesis. Lepidimoide with cytokinin stimulated the accumulation of chlorophyll and 5-aminolevulinic acid additively. In detached cucumber cotyledons, lepidimoide also promoted light-induced chlorophyll accumulation. These results indicate that lepidimoide, in cooperation with cytokinin, causes light-induced chlorophyll accumulation in the cotyledons of several dicot plant species by affecting the level of 5-aminolevulinic acid. Received April 4, 1997; accepted September 28, 1998     

Effects of wounding on cytokinin activity in cucumber cotyledons

Three known physiological responses to exogenous cytokinins were measured in wounded and nonwounded cotyledons from cucumber (Cucumis sativus L. cv Marketer) seedlings grown in darkness. Enhanced cell division, chlorophyll formation, and cotyledon expansion were detected in wounded cotyledons. The data suggest that wounding enhances endogenous cytokinin activity.     

Cytokinins and the Growth Responses of Seedlings of Betula pendula Roth. and Acer pseudoplatanus L. to Nitrogen and Phosphorus Deficiency

Deficiencies in the supply of nitrogen and phosphorus broughtabout the cessation of shoot extension and the formation ofterminal resting buds in seedlings of Acer pseudoplatanus maintainedunder long days, whereas seedlings of Betula pendula maintainedunder the same conditions continued extension growth of themain axis over a long period, although they showed symptomsof nitrogen deficiency, including a reduced chlorophyll content.The endogenous cytokinin levels of the base/nucleoside fractionin leaves of B. pendula were significantly lower in the nitrogen-deficientplants than in the controls only 3 d after transfer to deficientconditions. Similar, though less marked, changes were observedin response to phosphorus deficiency. Changes in the levelsof cytokinin activity in the nucleotide fraction in responseto nitrogen deficiency were much less marked. No significantchanges in cytokinin levels of the base/nucleoside or nucleotidefractions could be detected in leaf material from seedlingsof A. pseudoplatanus, even after a nitrogen or phosphorus deficiencytreatment of 4 weeks. Application of exogenous benzyladenineto seedlings of B. pendula subjected to nitrogen deficiencyresulted in a rapid regreening and to outgrowth of inhibitedlateral buds after only 2 d.     

The Influence of Atrazine, Benzyladenine, and the Embryonic Axis on Chlorophyll Synthesis in Cucumber and Squash Cotyledons

Excision of the embryonic axis prior to 3 1/2 days of germination in the dark followed by 8-h of light decreased the total chlorophyll content of cucumber cotyledons but not squash cotyledons. Benzyladenine stimulated the accumulation of chlorophyll in the cotyledons of intact embryos and excised cotyledons in both cucumber and squash. Gibberellic acid had no effect. Atrazine inhibited chlorophyll formation in excised squash cotyledons. Benzyladenine also increased the carotenoid and xanthophyll content in the cotyledons from intact squash seedlings. The results suggest that pigment synthesis in cotyledons may be controlled by a number of substances produced in the embryonic axis and that cytokinin-like benzyladenine can simulate the action of one of them.     

Effect of Hypocotyl Excision on Red Light-Mediated Betacyanin Formation in Amaranthus Seedlings

The effect of hypocotyl excision on red light-mediated betacyanininduction in cotyledons of Amaranthus caudatus L. was studiedusing etiolated, 3-day-old half-seedlings and isolated cotyledons.The removal of the hypocotyl promoted betacyanin formation undersafelight conditions in a manner competitive with brief (5 mins),but additive with prolonged (6 h) red illumination. If a papersupport was provided in order to improve the aeration, betacyaninformation in safelight conditions was further stimulated, reducingthe inductive effect of brief, but not changing the action ofprolonged red illumination. These results demonstrate that betacyaninphotoregulation is restricted to cotyledons of A. caudatus seedlings,with no evidence for transmission of light signals between differentorgans. Excision and aeration appear to promote selectivelya very low fluence response (VLFR) induced by safelight, suggestingdifferent mechanisms of phytochrome phototransduction underVLFR, low fluence response induced by brief saturating red light(LFR) and high irradiance reaction (HIR) occurring under prolongedred illumination. Amaranthus caudatus L, betacyanin photoregulation, red light, very low fluence phytochrome reaction     

Stimulation by Ethylene of Chlorophyll Biosynthesis in Dark-grown Cucumber Cotyledons

Five-day-old etiolated cucumber (Cucumis sativus L. var. Alpha Green) cotyledons produced more chlorophyll over a 4-hour illumination period after a prolonged exposure (12 to 72 hours) in the dark to ethylene concentrations ranging from 0.1 to 10 μl/l. Intact seedlings and excised cotyledons responded in the same way to this treatment. This effect does not involve a shortening of the lag phase of chlorophyll accumulation. Exposure of cotyledons to ethylene during the illumination period did not produce the same stimulatory effect on chlorophyll synthesis and, under certain conditions, chlorophyll synthesis was slightly inhibited by exposure to ethylene in the light.     

Enzymatic changes in gourd and bean cotyledons during ageing and the effect of detopping

Cotyledons of gourd (Cucurbita maxima Duchesne) and bean (Phaseolus vulgaris L.) were used to study the changes in the activities of catalase, peroxidase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase during ageing and the diversion in such changes that occur when cotyledon senescence was retarded by detopping the seedlings above the cotyledons. Catalase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase activities declined during the senescence of the cotyledons. When cotyledon senescence was retarded by detopping as marked by the increase in the levels of chlorophyll and protein, there was also an increase in the activities of these enzymes. Peroxidase activity, on the other hand, increased during the senescence of the cotyledons and detopping the seedlings resulted in a further increase in the peroxidase activity. It can be suggested that some root factor(s) probably cytokinin(s) is (are) mobilised into the cotyledons of the detopped seedlings which otherwise would have been mobilised into the shoot apices, and help retard or even reverse the senescence of the cotyledons.     

Phytohormonal changes in greening and senescing intact cotyledons of oilseed rape and pumpkin: influence of the growth retardant BAS 111 ˙˙ W

The triazole growth retardant BAS 111‥W delayed senescence in cotyledons of pumpkin ( Cucurbita maxima L. cv. Gelbe genetzte Riesenmelone) and stimulated chlorophyll synthesis in greening cotyledons of oilseed rape ( Brassica napus L. cv. Petranova) seedlings. In both cases, changes of phytohormone-like substances in the cotyledons were analyzed on a fresh weight basis by immunoassay.
After soil treatment with increasing retardant concentrations, a close correlation was observed in senescing cotyledons of pumpkin between a reduced loss in total chlorophyll and increasing levels of dihydrozeatin riboside (DZR) and trans -zeatin riboside (ZR)-type cytokinins. In contrast, the levels of isopentenyladenosine (IPA)-type cytokinins, 3-indoleacetic acid (IAA) and gibberellin (GA) did not change significantly. The levels of abscisic acid (ABA) were slightly elevated at low retardant concentrations but dropped considerably below those of controls at higher doses. Consequently, the molar ratio of total cytokinin to ABA content changed from approximately 1:40 in controls (50% of initial chlorophyll) to 1:3 in cotyledons treated with 3 mg BAS 111‥W plant⁻¹ (85% of initial chlorophyll). These changes, together with the known reduction of ethylene production by plants treated with nitrogen-heterocyclic retardants, can explain the delayed senescence in pumpkin cotyledons. Likewise, when etiolated, BAS 111‥W-treated seedlings of oilseed rape were exposed to light, the stimulation of chlorophyll synthesis in the cotyledons was accompanied by an accumulation of DZR- and, particularly, ZR-type cytokinins and IAA. In contrast, GA and ABA contents decreased slightly. We conclude that the influence of BAS 111‥W on cytokinin levels might be involved in the stimulation of greening.     

Stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanone] changes the morphology and partitioning of dry matter in cucumber ( Cucumis sativus L. cv. National Pickling) seedlings. The dry weights, potassium and cytokinin levels in the cotyledons and roots of the treated seedlings were higher, whereas the hypocotyl weights were lower than the controls. When etiolated intact seedlings or cotyledons excised from triadimefon-pretreated dark-grown seedlings were exposed to light, chlorophyll synthesis in the pretreated cotyledons was stimulated. Triadimefon does not have cytokinin-like activity in the cucumber cotyledon greening bioassay, but appears to induce the plants to produce more cytokinims, probably by stimulating root growth. Hence it is proposed that the stimulation of chlorophyll production by triadimefon in cucumber cotyledons is mediated by maintaining high levels of potassium and cytokinins in the cotyledons.     

Chloroplast development in 4-thiouridine-cultured radish seedlings III. Photochemical activities in isolated plastids

Accumulation of chlorophyll, development of photosystem I andII activities and contents of chloroplastic components wereinvestigated in greening radish seedlings germinated and grownwith 4-thiouridine (4SU). The development of photosystem I activityprior to that of photosystem II was observed also in the 4SU-culturedgreening radish cotyledons in which chlorophyll accumulationwas inhibited up to 60–80% of that of the control. Photochemicalactivities expressed on a plastid protein basis decreased withthe increase of 4SU in the culture medium. In contrast to ferredoxinand ferredoxin-NADP reductase, which were present in significantamounts in the treated cotyledons, chloroplastic cytochromes(f, b₅₅₉ and b₆ decreased in the plastids from 4SU-culturedcotyledons. These results suggest that 4SU interferes in partwith protein synthesis in plastids and thereby with chloroplastdevelopment. (Received December 4, 1979; )     

External calcium requirements for light induction of chlorophyll accumulation and its enhancement by red light and cytokinin pretreatments in excised etiolated cucumber cotyledons

Excised etiolated cucumber (Cucumis sativus L.) cotyledons that were depleted of external Ca²⁺ by equilibration with a Ca²⁺ buffer, which maintained the free Ca²⁺ concentration at 10^–8 M, failed to accumulate chlorophyll upon a 2-h exposure to white light. Increasing amounts of chlorophyll accumulation occurred at increasing external Ca²⁺ concentrations within the range of 10^–7-10^–3 M. Preillumination with red light or pretreatment with benzyladenine, which enhanced the rate of light-induced chlorophyll accumulation in control cotyledons, did not overcome the block to light-induced chlorophyll accumulation caused by the depletion of external Ca²⁺. Etiolated cotyledons that were treated with the Ca²⁺ ionophore, A23187, and then equilibrated with 10^–5 M Ca²⁺, accumulated significantly more chlorophyll during exposure to light than did untreated cotyledons. The enhancing effect of A23187 was approximately equal to that caused by red-light pretreatment. Etiolated cotyledons that were exposed to the Ca²⁺ channel-blocking agent, Nd³⁺ (neodymium), in the presence of 10^–5 M Ca²⁺, did not exhibit an enhancement of chlorophyll accumulation by red-light pretreatment, although they accumulated control levels of chlorophyll upon exposure to light and showed control levels of enhancement of chlorophyll accumulation by cytokinin pretreatment. Conversely, etiolated cotyledons that were equilibrated with 10^–5 M Ca²⁺ in the presence of nifedipine, a blocker of some Ca²⁺ channels, did not exhibit an enhancement of chlorophyll accumulation by cytokinin pretreatment, although they accumulated control levels of chlorophyll upon exposure to light and showed control levels of enhancement of chlorophyll accumulation by red-light pretreatment. These results indicate that external Ca²⁺ is required for chlorophyll accumulation by excised etiolated cucumber cotyledons during the first 2 h of light exposure, and that an influx of external Ca²⁺ is required for the enhancing effect of redlight and cytokinin. The differential abilities of Nd³⁺ and nifedipine to block the effects of red-light and cytokinin pretreatments suggests that enhancement of chlorophyll accumulation by red-light and cytokinin may involve different classes of Ca²⁺ channels.Abbreviations A23187 antibiotic 23187 calcium ionophore - Chl chlorophyll - nifedipine 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester We thank Randy Wayne for advice and encouragement.     

Synthesis and Turnover of {alpha}-Amylase in Cotyledons of Germinating Vigna mungo Seeds: Effects of Exogenously Applied End-Products and Plant Hormones

Pulse-chase experiments indicated that the higher levels ofa-amylase in detached and incubated cotyledons of Vigna mungothan those in cotyledons attached to the embryonic axis weredue to both faster synthesis and slower degradation of the enzymein the detached cotyledons than in the attached cotyledons.Levels of a-amylase in the cotyledons were examined in termsof possible effects of end-products and the effects of exogenouslyapplied plant hormones and growth regulators. Levels of a-amylaseactivity and content were reduced by high concentrations ofglucose and sucrose, and it is suggested that this effect wascaused mostly by osmotic stress and partly by end-product repression.The level of a-amylase was nearly twice that in controls after1 to 10µM GA₃ had been applied to the cotyledons. In addition,0.1 mM kinetin, 0.1 mM 2,4-D and 0.1 to 0.S mM naphthaleneaceticacid also increased the level by 34% to 66% as compared to thecontrol. ABA and uniconazole both prevented the synthesis ofa-amylase. (Received July 4, 1994; Accepted November 14, 1994)