Changes in Levels of Cytokinins in Etiolated Squash Seedlings after Illumination

When etiolated seedlings of squash (Cucurbita maxima Duch. cv.Houkou-aokawaamaguri) were exposed to light, their cotyledonsbegan to accumulate chlorophyll at a low rate for the firsthour but at a high rate therafter. After 0, 1 and 2 h of illumination,cytokinin levels in cotyledons, hypocotyls and roots were analyzedby ELISA (enzyme-linked immunosorbent assay) and GC-SIM (gaschromatography-selected ion monitoring). Cytokinin levels measuredby ELISA were about 10 times higher than those measured by GC-SIM,suggesting that ELISA was not suitable for the measurementsof cytokinin levels in the extracts from these tissues. Cytokininsfound by GC-SIM were t-RZ (t-ribosyl zeatin), c-RZ, t-Z (t-zeatin),c-Z, RiP (ribosyl isopentenyladenine) and iP (isopoentenyladenine).Levels of biologically active cytokinins (t-RZ and t-Z) didnot show marked changes after illumination. Levels of c-RZ andc-Z did, however, decrease in cotyledons and increase in hypocotylsafter illumination. Promotion of the accumulation of the chlorophyllin detached squash cotyledons by exogenously applied t-Z waspartially inhibited by the addition of c-Z, suggesting thatthe decrease in the endogenous level of c-Z in cotyledons ofintact seedlings after illumination permits the light-inducedformation of chlorophyll. (Received August 8, 1990; Accepted April 4, 1991)     

Irreversible Effects of Water Stress on Growth and Stomatal Development in Cotyledons of Etiolated Squash Seedlings

Growth of etiolated squash cotyledons and hypocotyls was suppressedwhen the seedlings were subjected to 60 mM polyethylene glycol(PEG) in 1/5 strength of Hoagland solution. The fresh weightof the hypocotyl completely recovered when the water stresswas relieved after one day of PEG treatment. The fresh weightof the cotyledons, however, did not completely recover eventhree days after the relief of water stress. The transpiration rate of the cotyledons was substantially reducedby the water stress, and it also did not completely recoverafter the water stress was relieved. Microscopic observationof stomata of the cotyledons by a replica method revealed thatthe water stress reduced the increase in both stomatal widthand density, and this reduction did not completely recover afterthe water stress was relieved. After one day of water stress, the endogenous ABA content ofthe cotyledon was increased from 68 to 114 ng/g fr wt. Afterthe water stress was relieved, the increased ABA content decreasedlinearly over two days to 20 ng/sg fr wt, the same value asin unstressed cotyledons. These results indicate that the effect of water stress on thehypocotyl growth was reversible but that the effect on the cotyledonswas irreversible. The irreversible effect of water stress onthe growth of the cotyledons probably resulted from the inhibitionof stomatal development, but endogenous ABA did not appear toinhibit the development. (Received March 24, 1986; Accepted June 25, 1986)     

Change in Nitrate-Reducing Activity in Squash Seedlings with NO2 Fumigation

Nitrogen dioxide (NO₂) fumigation inhibited nitrate reductase(NR, EC 1.6.6.1 [EC] ) activity assayed by an in vivo system in thecotyledons, but not in the first leaves, of squash (Cucurbitamaxima Duch.) seedlings. The inhibition was recovered when theseedlings were transferred to NO₂-free conditions, indicatingthat the effect of NO₂ was reversible. The NADH content in thecotyledons, photosynthetic O₂ evolution and respiratory O₂ uptakedid not change notably under NO₂ fumigation. Nitrate contentsin the cotyledons and first leaves did not change with NO₂ fumigation,but nitrite, ammonium and rapidly-metabolized amino acids contentsincreased. The inhibitory effect of NO₂ was also observed inthe in vitro assay, though the inhibition rate was smaller thanthat in the in vivo assay. These results indicate that the inhibitoryeffect of NO₂ on NR activity in squash cotyledons was derivedin part from the decrease in the amount of active NR due toammonium and/or amino acids accumulated in the tissue underNO₂ fumigation. (Received February 12, 1985; Accepted May 27, 1985)     

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)     

Occurrence of Indoleacetaldehyde and Tryptophol in the Extracts of Etiolated Shoots of Pisum and Helianthus Seedlings

Presence of a non-acidic, growth-promoting indole compound, identical in R_f and colour reaction and closely similar in UV spectrum to synthetic indoleacetaldehyde (IAAId) has been demonstrated in extracts of etiolated shoots of Pisum and Helianthus. On chemical as well as enzymic oxidation of this neutral extract, activity passes over into the acidic fraction. This neutral substance is believed to be IAAId and occurs in the free state as short-term, low temperature extractions indicate. On the basis of R_f, colour reaction and UV spectrum, tryptophol has also been identified in Helianthus extracts. Its identification in pea extracts, however, rests only on R_f, colour reaction and inactivity in Avena bioassays.     

玉米黄化幼苗生理生化特性的研究

以玉米品种'户单26'为材料,研究了黑暗条件下生长的玉米黄化幼苗内光合色素、可溶性蛋白含量和抗氧化酶活性的动态变化.结果表明,与正常条件下生长的对照组相比,黄化幼苗的叶绿素a、叶绿素b和类胡萝卜素含量均明显降低,特别是叶绿素a含量大幅度降低;黄化幼苗的抗氧化酶SOD、POD和CAT活性显著提高,可溶性蛋白含量也显著增加.研究发现,黑暗条件下的玉米幼苗叶绿素a的合成受到了显著影响而表现出黄化现象,黄化幼苗能主动提高其自身抗氧化酶活性和可溶性蛋白含量,减轻黑暗逆境对植物细胞的伤害.     

Studies on Greening of Etiolated Seedlings

Evidence is given that a selective light-pretreatment of the embryonic axis exerts a deep influence on the greening in primary leaves of 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Limburg). After a subsequent dark incubation of sufficient length and a final exposure of the entire plants to continuous illumination the lag phase of chlorophyll synthesis is completely removed. In particular the highly meristematic hook tissue seems to be responsible for this light effect. Lengthening of the dark period following pre-irradiation increased the capability of chlorophyll production in the main white light period, reaching its maximum after about 12 hours of darkness. The period of dark incubation for elimination of the lag phase is considerably longer in plants with shielded leaves than the length of the lag phase in etiolated seedlings of the same age, exposed entirely to continuous light. This difference may be explained by the synergistic effect between leaves and embryonic axis. Evidence for this interorgan cooperation is given by experiments with a selective light-pretreatment of leaves and embryonic axis. After a 5 min pre-exposure to white light of whole plants the leaves of some of the plants were shielded and these plants received a further pre-illumination of 2 hours on their embryonic axis. In all the pre-irradiated, etiolated plants the lag phase of chlorophyll synthesis was eliminated during the main white light period, following a dark incubation of 2 hours. Additional and preferential light activation of the embryonic axis during the pretreatment had no significant effect on chlorophyll production during the white light illumination after a 2 hours dark incubation, but resulted in a lower yield of chlorophylls after 18 hours dark incubation compared to the white light controls, receiving no selective light-pretreatment on the embryonic axis. From our results we can decisively conclude that a simultaneous light-pretreatment of both, leaves and embryonic axis, is more effective and beneficial for building up a capacity of chlorophyll synthesis in the leaves than either a selective light-pretreatment of the embryonic axis alone or a simultaneous pre-illumination of leaves and embryonic axis, immediately followed by an additional preirradiation of the embryonic axis. Therefore, we think that several photoactive sites are involved in de-etiolation processes of intact, etiolated seedings. Light activation of the embryonic axis stimulates the development of this organ and contributes to the greening processes in the leaf. At the same time, by irradiating the leaf, light activates the photo-sensitive site in the leaf itself, which also develops a capacity for chlorophyll synthesis. Both photo-acts are cooperative, explaining the enhanced chlorophyll production. Additional pre-irradiation of the embryonic axis after a short illumination of whole plants favours its own development and reduces the synthetic capacity of the leaf. A prolonged far-red pretreatment induces qualitatively the same response as white light. We assume that these effects on lag phase removal and chlorophyll production, induced in etiolated, primary bean leaves by selective irradiation of the embryonic axis, is a phytochrome-mediated process. Our results indicate a transmission of light-induced stimuli from one organ to another.     

Effect of Water Stress on Photochemical Activity of Chloroplasts during Greening of Etiolated Barley Seedlings

Water stress retards accumulation of chlorophyll and chlorophylla/b protein complex during greening of barley seedlings in light.The rate of 2,6-dichlorophenol indophenol (DCPIP) photoreductionin isolated chloroplast which decreases under water stress isenhanced significantly in the presence of electron donors, diphenylcarbazide (DPQ) and Mn²⁺. Under water stress, the decrease ofthe rate of oxygen evolution measured in continuous white lightwas 40–73% and that of oxygen uptake (as a measure ofelectron transport through PS I from reduced DCPIP) was onlyabout 20%. During greening, under water stress, (i) a differentialinhibition of PS II biosynthesis as compared to PS I occurs,(ii) the site of electron donation by DPC seems to be closerto the reaction center ofPS II, (iii) the oxidizing side ofPS II near the oxygen-evolving system is affected maximallyby water stress. (Received March 11, 1980; Accepted November 13, 1980)     

四季豆幼苗中尿囊酸酰胺水解酶的一些特性

酰脲代谢在许多固氮豆科植物氮素代谢中起重要作用;尿囊酸的酰胺水解酶(EC3.5.3.9)分解尿囊酸成为脲基乙醇酸和CO2、NH3,脲基乙醇酸的酰胺水解酶进一步分解脲基乙醇酸产生乙醛酸和CO2、NH3.该文首次报告测定四季豆尿囊酸降解酶(分解尿囊酸的酶)的方法,酶反应基质需要盐酸苯肼存在.在四季豆干种子、幼苗根、茎和叶,均可测出尿囊酸降解酶活力.从四季豆幼苗分离出两个尿囊酸降解酶.一个分子量大于200 kD,另一个分子量为13.5 kD;小分子量的尿囊酸降解酶(没有脲基乙醇酸酰胺水解酶或脲酶活力)用于性质研究.酶反应产物分析表明,该酶是尿囊酸的酰胺水解酶.该酶反应的最适pH为8.5.Mn2 是该酶的金属辅助因子.Km为76μmol/L,Vmax为16.7 nKat/mg(=1 002 nmol min1mg1).乙醛酸和乙醇酸抑制该酶活力.赖氨酸残基和色氨酸残基是酶活力的必需基团;巯基和酪氨酸残基不是酶活力的必需基团.     

Binding Proteins to Phytochrome A in Etiolated Pea Seedlings

In order to detect and characterize a putative receptor(s) fora signal from PhyA, proteins that bind to purified pea PhyAwere searched for in the crude extract of etiolated pea seedlingswith affinity chromatography. PhyA was coupled to the columnsubstrate either in P_R form (P_R column) or in red-irradiatedform (P_FR column). The coupled PhyA of both columns retainsits spectral reversibility between P_R and P_FR, although theirpeptide mapping by trypsin digestion suggests that the C-terminalhalf of PhyA in the P_FR column is partially fixed in P_FR structure.15 polypeptides were detected reproducibly in the elution fromthe P_FR column by silver-staining of SDS-PAGE. These 15 polypeptidesmay form two complexes judging from their elution profiles.Of the 15 polypeptides, the 6 major polypeptides have approximatemol wt of 80, 55, 53, 46, 40 and 35 kDa. On the other hand,only a trace amount of protein, which mainly consists of the46 kDa species, was eluted from P_R column, indicating the presenceof P_FR-specific BPs in the crude extract of etiolated pea seedlings.Of the 6 major polypeptides, the 40 kDa species binds to thePhyA in a photoreversible manner. (Received June 19, 1998; Accepted December 19, 1998)     

表油菜素内酯对离体黄化小麦叶片展开的影响

油菜甾醇内酯类化合物对叶片展开(leaf-unrolling)的生物活性,以表油菜素内酯效应为最强。epiBR的作用与其浓度及苗龄有关,0.0001ppm时已表现促进作用,至1ppm达最高;epi-BR对4d龄叶片促进最明显,随苗龄增加逐渐下降,表明幼嫩叶片比老叶片对epiBR更为敏感。epiBR能克服环己酰亚胺和放线菌素D对叶片展开的抑制;epiBR与脱落酸之间亦存在拮抗,而与6BA则表现加成作用。     

Photoregulation of beta-Tubulin mRNA Abundance in Etiolated Oat and Barley Seedlings

The effect of light on the abundance of β-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L. seedlings. Slot blot analysis employing an oat β-tubulin cDNA clone was used to measure β-tubulin mRNA levels. White light induced a 45% decrease in oat β-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in β-tubulin mRNA levels in oats and barley, respectively. Recovery of β-tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat β-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in β-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the β-tubulin genes.     

A Study of the Growth and Photoperceptivity of Etiolated Oat Seedlings

The photoperceptivity of intact oat seedlings has been observedafter pretreating them for 3 days in darkness with a numberof substances—copper and manganese ions, EDTA, copper-glycine,glycine, nitrates, indoleacetic acid, ethanol, and carbon dioxide. None enhanced photosensitivity; carbon dioxide and ethanol reducedit. Copper-glycine in darkness promoted mesocoty1 growth and inducedroot formation. The effects of carbon dioxide and ethanol are shown to be identicaland to account for this a speculative proposal is made aboutmaturation. An alternative explanation of the growth of the mesocotyl thenarises. The relationship between maturation and the behaviour of theseedling upon illumination is discussed.     

Roles of Abscisic Acid and Indoleacetic Acid in the Stunted Growth of Water-Stressed, Etiolated Squash Hypocotyls

The endogenous contents of indoleacetic acid (IAA) and abscisicacid (ABA) were assayed simultaneously in etiolated squash hypocotylsthat had been subjected to water stress applied to squash rootsby immersing them in 60 mM polyethylene glycol solution. Thegrowth rate and suction force of the hypocotyl under this water-stresscondition were determined for comparison with the two hormonecontents. Water stress retarded hypocotyl growth and increased the suctionforce of hypocotyl cells, as the ABA content increased, by 5-foldof the initial content one day after treatment with polyethyleneglycol. The ABA contents thereafter remained high up to day4. The increase in ABA content was detected in stressed hypocotylas early as 6 h after treatment, when water stress had not affectedhypocotyl growth. When the water stress was terminated one dayafter treatment began, hypocotyl growth immediately recovered,and the ABA content decreased to the value for unstressed hypocotylsin one day. The IAA content gradually decreased in unstressedhypocotyls, but was maintained at a high value in stressed hypocotyls. Logarithmic concentrations of endogenous ABA (ng/g fr wt) werecorrelated significantly with the suction force of the hypocotyls(r=0.92) and with the growth rates (r=–0.78). IAA contentwas correlated neither with the growth rate nor the suctionforce. These results suggest that the endogenous ABA contentwas associated with the stunted growth of the etiolated squashhypocotyls produced by water stress but that the endogenousIAA content did not have an important role in growth regulationunder water stress conditions. (Received April 11, 1984; Accepted October 2, 1984)