Auxin-induced extension of the isolated epidermis of light-grown pea epicotyls

1. The effect of IAA and FC on the extension of isolated epidermisof light-grown Alaska pea epicotyls was studied under differentconditions with an extension apparatus. The following resultswere obtained.
2. The epidermis extended in response to low pHbuffer solutionof 1–10 mM, maximum extension being achievedat pH below5.5.
3. IAA, 5 mg/liter, caused, although not consistently,an extensionof epidermal strips in 1 mM buffer, but not at10 mM.
4. Consistent extension of the isolated epidermis dueto IAA wasobtained by addition of GTP, ATP, ITP or UTP (sodiumsalts),but not nucleosides, nitrogen bases or sugars.
5. A fungaltoxin, FC, at 10^–5 M induced extension of theepidermiswithout addition of the nucleoside triphosphates.
6. IAA andFC caused H⁺ extrusion in peeled epicotyl segments bothin thepresence and absence of GTP. IAA caused appreciable H⁺extrusionin the isolated epidermis only in the presence ofGTP, whereasH⁺ extrusion by the epidermis was induced by FCeven in theabsence of GTP.

From these results, we concluded that IAA induces extensionof the isolated epidermis under the above conditions throughthe mediation of H⁺ ions. (Received July 12, 1976; )     

Auxin and hydrogen ion actions on light-grown pea epicotyl segments III. Effect of auxin and hydrogen ions on stress-relaxation properties

The effect of auxin and hydrogen ions on the stress-relaxationproperty of the cell wall was studied using light-grown peaepicotyl segments. Auxin pretreatment caused a decrease in theminimum relaxation time (T_o) of the cell wall of stem segmentsand the epidermis at different pH values, when auxin had beengiven to stem segments. The in vivo effect of hydrogen ionson the cell wall was found to be the same as that of auxin.The stress supported by the frozen-thawed epidermis relaxedfaster at pH 4.5 than at pH 6.5, resulting in a shorter maximumrelaxation time (T_m) at pH 4.5. This effect of low pH was inhibitedby low temperature. The incipient pH range producing this effecton Tm was found to be 5.2–5.8. (Received July 22, 1974; )     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

The Inhibition of Growth by Gravitropic Stimulation in Darkness in Agravitropic Primary Roots of Zea and the Role of Calcium

The primary roots of the "Golden Cross Bantam 70" cultivar ofZea mays are agravitropic in darkness and their orthogravitropismis light-dependent. Analysis of the agravitropic roots providesimportant information about the mechanism of orthogravitropism.However, the underlying mechanism of the agravitropic responsein darkness is unknown. We found that the growth of intact primaryroots was inhibited by gravitropic stimulation (i.e., changingthe orientation of the roots from vertical to horizontal) indarkness, but that of detipped roots was not. The role of calciumin this gravistimulation-dependent inhibition of growth wasinvestigated using apical 5-mm segments of the primary roots.The gravistimulation-dependent inhibition of growth was preventedby applying 10 mM MES-KOH buffer at pH 6.0 to the root cap.By contrast, the application of 0.1–1 mM buffer at pH6.0 and 10 mM buffer at pH 4.5–5.0 allowed the gravistimulation-dependentinhibition of growth. Furthermore, when the buffer of 10 mM(pH 6.0) contained 1–5 mM CaCl₂, the gravistimulation-dependentinhibition of growth was apparent. By contrast, when weak (1mM) buffer at pH 6.0 or 10 mM buffer at pH 4.5 contained 5 mMEGTA, no gravistimulation-dependent inhibition of growth wasobserved. Thus, the gravistimulation-dependent inhibition ofgrowth in darkness seemed to be mediated by an increase in thelevel of free Ca²⁺ in the root tip. These results suggest thatfree Ca²⁺ in the apoplast of the root tip plays an importantrole in the agravitropic response in darkness as well as inorthogravitropism under light of the roots of this cultivarof Zea mays. (Received March 21, 1994; Accepted July 25, 1994)     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

A simple method for the isolation of intact mesophyll protoplasts from cereal plants

Intact mesophyll protoplasts from cereal plants were easilyprepared by incubating leaves with the abaxial epidermis peeledoff at 20–25?C for 2–3 hr in 0.6 M mannitol containing1% cellulase at pH 5.6. From one gram (fresh weight) of leaves1.5–6?10⁶ protoplasts, more than 90% of which were morphologicallyintact, could be obtained. Protoplasts isolated from wheat,oat, corn and barley were efficiently infected with brome mosaicvirus (BMV), and supported viral multiplication. (Received June 21, 1977; )     

Effect of osmotic shock on auxin-induced cell extension, cell wall changes and acidification in Avena coleoptile segments

The effect of plasma membrane alteration caused by osmotic shockof different strengths on the auxin-induced responses of Avenacoleoptile cells was observed. Osmotic shock brought about by0.5–0.7 M mannitol solution for 10 or 30 min, followedby phosphate-buffer (1 mM, pH 6.0) treatment for 10 min at 4?Ccaused no significant inhibition of auxin-induced cell extension.The osmotic shock did not affect auxin-induced cell wall looseningrepresented by stress-relaxation time and a decrease in thenoncellulosic glucose level of the cell wall. The shock causedonly a temporary inhibition of transmembrane potential and noinhibition of oxygen consumption. However, it inhibited auxin-stimulatedH⁺ secretion which was reversed by 0.1 mM CaCl₂. We concludedthat the Osmotic shock may partly modify the plasma membranerelated to the hydrogen ion pump which interacts with auxin,but this modification which is reflected little by the transmembranepotential and cellular metabolism, is not closely related toauxin-induced cell wall loosening and thus cell extension inAvena coleoptiles. ³ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received February 17, 1978; )     

Auxin and hydrogen ion actions on light-grown pea epicotyl segments I. Tissue specificity of auxin and hydrogen ion actions

Growth responses to added auxin and hydrogen ions of differentlyprepared tissue segments excised from 5th internodes of light-grownAlaska peas were studied. Unpeeled segments extended in responseto auxin but not to hydrogen ions. Peeled segments elongatedwell in response to hydrogen ions but much less when exposedto auxin. Hollow-cylinder (central part removed) and infiltrated(by reduced pressure) unpeeled segments significantly elongatedin response to hydrogen ions as well as to auxin. Outward curvatureof split segments was enhanced by buffer solution at pH lowerthan 5.0. Peeled segments secreted hydrogen ions into an incubationmedium containing auxin or a fungal toxin, FC. Unpeeled segmentsand the isolated epimermis secreted much less hydrogen ionsin response to these agents. Hydrogen ions generated by theaction of auxin thus seem to accumulate in the region adjacentto the cuticle, that is, epidermal cells. The significance ofthe epidermis in auxin and hydrogen ion actions is discussed. (Received May 17, 1974; )     

Characterization of membrane-bound Mg++-activated ATPase isolated from the lower epidermis of tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase was separated from thelower epidermis of tobacco leaves (Nicotiand tabacum L. SamsunNN) on stepwise sucrose density gradient centrifugation. Membrane-bound epidermal ATPase was localized in the interfaceof densities in sucrose of 1.12 to 1.16 in the sedimentary fractionbetween 1,500?g to 10,000?g from the homogenate of the lowerepidermis. The epidermal ATPase activity was activated by divalentcations (Mg⁺⁺>Mn⁺⁺Co⁺⁺>Fe⁺⁺>Zn⁺⁺>Ca⁺⁺) and furtherstimulated by KCl by ca. 20%. The pH optimum for Mg⁺⁺-activationof the epidermal ATPase was ca. 6.0. The enzyme hydrolyzed ATPmore rapidly than other nucleoside triphosphates. The optimumtemperature for activation of the epidermal ATPase activitywas ca. 40?C. 50% of the epidermal ATPase activity was lostin 18 min at 55?C and in 2.5 days at 2.5?C. The apparent Kmvalue of the epidermal ATPase was 4.7?10^–4 M and Vmaxwas 65.4 nmoles Pi/mg protein/min. The epidermal ATPase wasstrongly inhibited by N, N'-dicyclohexylcarbodiimide (DCCD)in vitro whereas oligomycin, carbonyl cyanide m-chlorophenylhydrazone(CCGP), indoleacetic acid (IAA) and abscisic acid (ABA) wereinsensitive to the epidermal ATPase activity. (Received May 23, 1978; )     

FORMATION AND CHARACTERIZATION OF THREE TYPES OF YEAST INVERTASE

Three types of invertase (invertase I, II and III) are separatedfrom the soluble and insoluble fractions (4,500xg, 10 min supernatantand pellets of the homogenate, respectively) of baker's yeastby a DEAE cellulose column chromatography. The invertases Iand II are eluted with 0.1 M sodium acetate buffer (pH 3.9)and with 0.1 M sodium acetate buffer (pH 6.2) containing 0.1M NaCl from DEAE cellulose respectively, whereas the invertase-IIIremains adsorbed on the cellulose under these conditions. Theyare present in proportions of 2.5: 1 : 0.06 in the soluble fractionand 1.4: 1 : 0.12 in the insoluble fraction of the fresh baker'syeast cells. While in-vertase-II remains at a constant level,invertases I and III in the soluble fraction increase upon incubationof cells for the formation of invertase under the continuoussupply of sucrose. Invertases I and II differ from each other considerably in theoptimum pH and slightly in the response to (activation and inactivationby) crude papain and are identical with respect to the heatstability and probably to the affinity for sucrose. ¹Present address: Chemical Laboratory, Nippon Medical School,Konodai, Ichikawa-shi, Chiba-ken.     

Cell Wall Acidification and its Role in Auxin-Stimulated Growth

The role of cell wall acidification in auxin-stimulated growthwas examined in abraded hypocotyl segments of etiolated Cucumis.sativus seedlings. Acidification of the medium by these segmentswas strongly inhibited by a pretreatment and the continued presenceof 1?0 mol m^–3 vanadate, widely used as an inhibitor ofplasma membrane ATPase activity. Elongation of segments in pH6?5 buffer was almost completely inhibited by such a treatmentwith vanadate, and the promotion of growth by indole-3-aceticacid (IAA) seen in the absence of vanadate was completely abolished.However, both inhibited and uninhibited segments showed a pronouncedelongation in response to pH 4?0) buffer. In pH 4?0 buffer,in contrast to the results obtained at pH 6?5, IAA significantlypromoted growth in both the presence and absence of vanadate.The results indicate that IAA can promote growth in the absenceof endogenous acidification, but that an acid wall is necessaryfor wall loosening to occur. Key words: Acidification, auxin-stimulated growth, Cucumis sativus, vanadate     

Effects of KCI Concentration on Changes in Starch and Malate Levels in Abaxial and Adaxial Epidermis in Relation to Stomatal Opening

The effects of KCI concentration in the incubation medium onstomatal opening and starch and malate metabolism in isolatedabaxial and adaxial epidermis of Commelina communis were investigated.Increasing KCI produced progressively wide apertures, reducedstarch hydrolysis and malate synthesis, but failed to eliminatethe normal disparity in abaxial and adaxial opening. The fusicoccin-stimulatedwide opening was accompanied by a dramatic enhancement of malateformation and starch hydrolysis at zero KCI, but not at 200mol m^–3 KCI, indicating a controlling influence of KCIconcentration on carbohydrate and malate metabolism. Accordingly,malate and Cl^– appear to compete for K⁺, and the one assumesan increasingly decisive role as the other becomes limiting.However, starch hydrolysis and malate production must play acentral role in stomatal opening in intact leaves because theyare unlikely to have such high Cl^– regimes as 200 molm^–3, though not necessarily in isolated epidermis incubatedin media enriched with inorganic ions. The restricted adaxial opening is attributed to limited K⁺ accumulation,starch hydrolysis and malate production in the guard cells,but the primary cause is not known. Key words: Stomata, Abaxial/Adaxial Epidermis     

CdSe量子点荧光猝灭法测定尿嘧啶

以CdSe量子点为荧光探针,基于荧光猝灭法对碱基尿嘧啶进行了定量检测,考察了缓冲液体系、反应时间、量子点浓度等多种因素的影响. 实验结果表明,在pH 7.4的0.2 mol/L Na₂HPO₄-NaH₂PO₄缓冲液中,反应时间为60 min,尿嘧啶浓度为10^-6～10^-4mol/L范围时,其线性回归方程为F₀/F =0.992+3.35×10⁴Q (mol/L),检测限为3.23×10^-6 mol/L(即0.36μg/ml). 该方法检测范围宽,灵敏度高,为尿嘧啶的测定提供了新的方法.     

中国木犀科苦丁茶ISSR实验条件优化的研究

系统地研究了中国木犀科苦丁茶ISSR反应体系中的主要影响因子,建立了一套稳定的ISSR PCR反应参数。筛选出了10个有效引物,并以中国木犀科苦丁茶8个物种共21份种质材料为供试材料对优化后的反应条件的重复性、多态性进行了检测。优化后的反应体系为：10×buffer 2.5 μL,2.0~3.0 mmol·L^-1 MgCl₂,150~300 μmol·L^-1 dNTPs,Taq酶1.0~1.5 U,引物0.4~0.5 μmol·L^-1,DNA模板5~320 ng。PCR扩增程序为：94℃预变性4 min,然后按94℃变性40 s,50~54℃退火45 s,72℃延伸120 s,进行35个循环,最后72℃延伸8 min。该反应条件可应用于中国木犀科苦丁茶亲缘关系和遗传多样性分析。     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Freezing of rat lymphocytes. I. The effects of dimethyl sulfoxide and freezing on the phytohemagglutinin- and pokeweed mitogen-responding lymphocyte subpopulations.

Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide (DMSO) at 1 °C/min and stored at ?196 °C for 10 min. The functional recovery of the cell populations was monitored by the mitogenic response (uptake of [³H]thymidine) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozen-thawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures.A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than 10-fold higher than that present in the culture medium after freezing and thawing.Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations.     

Extensibility and rheology of collenchyma cells: II. Low-pH effect on the extension of collocytes isolated from high- and low-growing material

Low-pH effects were studied on the extension of isolated fresh,methanol-killed and frozen-thawed collocytes. Fresh and frozen-thawedsamples responded to low pH. This response decreased with increasingdifferentiation. A yield stress was found for frozen-thawedsamples. The significance of the response during growth anddifferentiation is discussed. (Received February 21, 1977; )     

百合鳞茎淀粉磷酸化酶提取条件的优化

为深入研究百合鳞茎淀粉磷酸化酶（SP）的性质及其在鳞茎淀粉代谢中的生理作用,本试验确定了兰州百合鳞茎内SP的最佳提取和测定条件,并对部分酶学性质进行了初步探索。结果表明：SP的最适提取缓冲液为pH5.8的琥珀酸缓冲液;最佳反应体系为以25 mmol·L^-1的Glc-1-P为底物,在30℃条件下反应10 min。在pH5~6范围内,SP活性最高;百合鳞茎的SP不易保存且不耐热,4℃条件下保存12 h,酶活力最高残留50%;40℃条件下保存30 min后,SP活力几乎完全丧失。     

The Light-Growth Response of Vaucheria. A conditio sine qua non of the Phototropic Response?

This is the first report of a positive light-growth response(LGR) in tip-growing cells. Tip-growth of a coenocytic fresh-wateralga Vaucheria terrestris was temporarily accelerated by a shortpulse of blue light. The LGR occurred after a lag period shorterthan 1 min, and reached its maximum ca. 3 min after the onsetof blue light. The growth rate then rapidly decreased againand often showed a damped oscillation with a period of about10 min. If the blue light pulse was shorter than 2 min, themagnitude of the LGR seemed to obey the reciprocity law. Anothertype of growth promotion, the apical expansion, is brought aboutwhen the pulse is longer than 5 min. In this report, however,only the LGR which is caused by a short pulse of blue light,is dealt with. The threshold fluence at 456 nm was ca. 10 Jm^–2 at pH 7.0. The response was very sensitive to thepH of the medium: it was 5 J m ^–2 at pH 7.5, 150 J m^–2at pH 6, and 750 J m^–2 at pH 5. The phototropic responsealso showed a very similar pH-dependency between pH 5.5 andpH 6.5. The relationship between the positive LGR and positivephototropic response was found to be much closer in this tip-growingalga than in diffuse-growing cells. The possibility that theLGR is the primary and essential process preceeding the phototropicresponse is discussed.     

Abscisic acid levels and metabolism in the leaf epidermal tissue of Tulipa gesneriana L. and Commelina communis L.

We have shown the presence of abscisic acid (ABA) in abaxial epidermal strips taken from Tulipa gesneriana and Commelina communis and that the ABA level rises in the epidermis when leaves are water stressed. ABA levels had risen 50% in the abaxial epidermis of C. communis 30 min after the leaves lost 10% of their fresh weight. Epidermis from both T. gesneriana and C. communis metabolize [¹⁴C]ABA to several products probably including phaseic acid (PA) and dihydrophaseic acid (DPA).Abbreviations ABA abscisic acid - RIA radioimmunoassay - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography