Age-Dependent Changes in Levels of Ascorbic Acid and Chlorogenic Acid, and Activities of Peroxidase and Superoxide Dismutase in the Apoplast of Tobacco leaves: Mechanism of the Oxidation of Chlorogenic Acid in the Apoplast

In order to understand browning in tobacco plants during aging,age-dependent changes in the levels of ascorbic acid (AA) andchlorogenic acid (CGA) and its isomers were investigated inthe apoplast and the symplast of the leaves. Also activitiesof peroxidase (POX) and superoxide dismutase (SOD) were determined.AA decreased during aging until it was no longer detectablein the apoplast, while symplastic AA remained although the leveldecreased on aging. In contrast, levels of CGA and its isomersand activity of POX in the apoplast increased on aging, whilethose in the symplast remained nearly constant in mature andold leaves. The activity of SOD in the apoplast increased duringaging, while that in the symplast decreased. Oxidation of CGAby the apoplastic solution was observed in the absence of externallyadded H₂O₂ and the oxidation was inhibited by SOD and catalase.Brown components, which contained caffeic acid moieties, accumulatedin the apoplast on aging and the components produced O–₂and H₂O₂ by autooxidation. From these results, we conclude (i)that brown components are formed in the apoplast by the CGA/POXsystem, (ii) that the H₂O₂ required for the reaction can beprovided by the CGA/POX system itself and by autooxidation ofthe brown components, and (iii) that apoplastic SOD functionsto generate H₂O₂ from apoplastically formed O–₂. (Received February 8, 1999; Accepted May 7, 1999)     

Regulation of Peroxidase-Dependent Oxidation of Phenolics in the Apoplast of Spinach Leaves by Ascorbate

The aqueous phase of the cell walls inside leaves (apoplast)of spinach contained ascorbate (AA) and dehydroascorbate (DHA).Ratios of AA to AA plus DHA were between 0.4 and 0.9, whereasthose inside leaves were higher than 0.9. The amounts of AAplus DHA in the apoplast were between 15 and 60 nmol (g fr wt)^–1of leaves. If the volume of the apoplast is about 10% of totalvolume of leaf cells, the concentrations of AA plus DHA werebetween 0.15 and 0.6 mM. Apoplastic AA was oxidized by hydrogenperoxide, and the oxidation was stimulated by phenolics suchas caffeic acid or ferulic acid by a factor of 10, suggestingthe presence in apoplast of peroxidases which are differentfrom AA peroxidase. The stimulation was due to the oxidationof AA by the primary oxidation products of phenolics with apoplasticperoxidase. Based on the data, the physiological significanceof the occurrence of AA in the apoplast is discussed in relationto the regulation of the apoplastic oxidation of phenolics. (Received January 8, 1992; Accepted February 28, 1992)     

Energized Uptake of Ascorbate and Dehydroascorbate From the Apoplast of Intact Leaves in Relation to Apoplastic Steady State Concentrations of Ascorbate

Abstract: Transport of ascorbate (AA) and dehydroascorbate (DHA) through the petiole into detached leaves of Lepidium sativum and other plant species via the transpiration stream, and energized uptake into leaf tissue, were measured indirectly by recording changes in membrane potential and apoplastic pH simultaneously with substrate‐stimulated respiration and transpiratory water loss. When 25 mM AA or DHA was fed to the leaves, steady state respiration at 25 °C was transiently increased by more than 50 % with AA and 70 % with DHA. Stimulation of respiration was accompanied by a transient breakdown of membrane potential followed by alkalinization of the leaf apoplast suggesting energized uptake at the expense of the transmembrane proton motive force. The average CO₂/AA ratio calculated from stimulated respiration during ascorbate uptake was 0.76 ± 0.26 (n = 17). The corresponding ratio for DHA was 1.38 ± 0.28 (n = 11). Far lower CO₂/substrate ratios were observed when NaCl or KCl were fed to leaves. The differences indicate either partial metabolism of AA and DHA in addition to energized transport, or less likely, higher energy requirement for transport of AA and DHA than for the inorganic salts. Maximum rates of energized AA transport into leaf tissue (deduced from maxima of extra respiration and calculated on the basis of CO₂/AA = 0.76) were close to 650 nmol m^‐2 leaf area s^‐1, i.e. far higher than most previously reported rates of transport. When the apoplastic concentration of AA was decreased below steady state levels during infiltration/centrifugation experiments, AA was released from leaf cells into the apoplast. This suggests that AA oxidation to DHA in the apoplast (as occurs during extracellular ozone detoxification) triggers energized transport of the DHA into the symplast and simultaneously AA release from the symplast into the apoplast, perhaps together with protons in a reversal of the energized uptake process.     

Pathway of phloem loading in the C3 tropical orchid hybrid Oncidium Goldiana

The apoplast of mature leaves of the tropical orchid OncidiumGoldiana was perfused with 0.5 mM p-chloromercuribenzenesulphonicacid (PCMBS) via the transpiration stream in order to test themode of phloem loading. The efficacy of introducing PCMBS byperfusion was shown by saffranin O dye movement in the veinsand leaf apoplast in control experiments. Photoassimilate exportas the result of phloem loading was measured by collection of¹⁴CO₂-derived photoassimilates from the basal cut-ends of intactleaves. Phloem loading and translocation of photoassimilates was inhibitedby 89% in leaves perfused with PCMBS for 1 h. The effect ofPCMBS on leaf photosynthesis was minimal. The amount of radiocarbonfixed by PCMBS-treated leaves averaged 89% of control leavesperfused with distilled water. A negative correlation betweenthe total amount of photoassimilate exuded and the calculatedconcentration of PCMBS in the leaf apoplast was also observed.The results indicate that phloem loading in Oncidium Goldianaoccurs via the apoplastic pathway. Key words: Phloem loading, apoplast, PCMBS, tropical orchid     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Redox state of ascorbic acid in the apoplast of stems of Kalanchoë daigremontiana

The aqueous phase of cell walls in stems of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie (apoplast) contained ascorbic acid (AA) and dehydroascorbic acid (DHA). Ratios of AA/(AA + DHA) were 0.31 ± 0.12 (SD, n = 4), whereas those of whole stems (tissues plus apoplast) were >0.9. The amounts of (AA + DHA) in the stems were 1970 ± 190 (SD, n = 4) nmol g⁻¹ fresh weight and those in the apoplast were 14 ± 2 (SD, n = 4) nmol g⁻¹ fresh weight of stems. Ratios of AA/(AA + DHA) differed in different tissues of the stems. The ratios of AA/(AA + DHA) of apoplast plus symplast were in the following order: pith ⋍ epidermis plus cortex > vascular bundle system, and those of apoplast were: pith > epidermis plus cortex > vascular bundle system. Ratios of AA/(AA + DHA) in the apoplast of the different tissues decreased to about 1/3 of the original values after wounding, while the amounts of (AA + DHA) remained largely unaffected. In contrast, soluble apoplastic peroxidase activities increased 30- to 70-fold on wounding. Hydrogen peroxide infiltrated into stems caused a rapid oxidation of AA. Coniferyl alcohol was oxidized by peroxidase in intercellular washing fluid and by cell wall-bound peroxidase. The oxidation of coniferyl alcohol by peroxidase in intercellular washing fluid was completely inhibited as long as AA was present in reaction mixtures. The oxidation of the coniferyl alcohol by cell wall-bound peroxidase was partially inihibited by AA and the degree of inhibition was dependent upon the concentration of AA. The possible functions of AA in the apoplast are discussed in relation to the control of peroxidase-dependent oxidation of phenolics.     

Apoplastic pH of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

Comparison of Palaeobotanical Observations with Experimental Data on the Leaf Anatomy of Durmast Oak [Quercus petraea(Fagaceae)] in Response to Environmental Change

To test whether stomatal density measurements on oak leaf remainsare reliable tools for assessing palaeoatmospheric carbon dioxideconcentration [CO₂], under changing Late Miocene palaeoenvironmentalconditions, young seedings of oak (Quercus petraea,Liebl.) weregrown at elevatedvs.ambient atmospheric [CO₂] and at high humiditycombined with an increased air temperature. The leaf anatomyof the young oaks was compared with that of fossil leaves ofthe same species. In the experiments, stomatal density and stomatalindex were significantly decreased at elevated [CO₂] in comparisonto ambient [CO₂]. Elevated [CO₂] induced leaf cell expansionand reduced the intercellular air space by 35%. Leaf cell sizeor length were also stimulated at high air humidity and temperature.Regardless of a temperate or subtropical palaeoclimate, leafcell size in fossil oak was not enhanced, since neither epidermalcell density nor length of the stomatal apparatus changed. Theabsence of these effects may be attributed to the phenologicalresponse of trees to climatic changes that balanced temporalchanges in environmental variables to maintain leaf growth underoptimal and stable conditions.Quercus petraea,which evolvedunder recurring depletions in the palaeoatmospheric [CO₂], maypossess sufficient phenotypic plasticity to alter stomatal frequencyin hypostomatous leaves allowing high maximum stomatal conductanceand high assimilation rates during these phases of low [CO₂].Copyright1998 Annals of Botany Company Atmospheric CO₂, high humidity, elevated temperature,Quercus petraea,durmast oak, Late Miocene, palaeoclimates, leaf anatomy, stomatal density, stomatal index     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Photosynthesis and Light Activation of Ribulose 1, 5-bisphosphate Carboxylase in the Presence of Starch

Owing to a typographical error three equations were omittedfrom page 1294. The correct paragraphs are set out below. The component K₁ corrected for the difference in temperaturebetween the enzyme assay and the leaf and was calculated accordingto the Arrhemus equation. where v10 and v18 are the reaction velocities of carboxylationat 10?C and 18?C, respectively and A is the activation energy(A = 90 kJ mol^–1, as determined for purified wheat RuBPCOby M?chler, Keys and Cornelius, 1980) The components K2 corrected for the difference in CO₂ partialpressure between enzyme assay and leaf and for competitive inhibitionof carboxylation by O₂ and was calculated according to the modifiedMichaelis Menten equation where v_c, is the carboxylation velocity under leaf conditions,V_c. is the maximum carboxylation velocity as determined in theenzyme assay, K_c, and K_o are the Michaelis constants for carboxylationand oxygenation, respectively (K_o = 159 Pa CO₂. K_o = 35.3 kPaO₂, as interpolated for 18?C from spinach data as determinedby Jordan and Ogren, 1984), O is oxygen partial pressure inair and C₁ is intercellular CO₂ partial pressure in leaves (C₁= 29.1 ? 0.8 Pa (? s c , n = 15)) The component K3 corrected for the decrease in CO₂ fixationin leaves due to photorespiration and was calculated accordingto equation 3 Equation 3 is denved from the equation for the substrate specificityof RuBPCO, S= v_c/v_oC (Laing, Ogren, and Hageman, 1974), andfrom the equation for the stoichiometry of photorespiratoryCO₂ release, F=v_c–1/2 v_o, where v_c, and v_c are reactionvelocities of carboxylation and oxygenation, O and C are partialpressures of 02 and intercellular CO₂, F is net photosynthesisand S is the substrate specificity of RuBPCO (S= 3061 Pa/Pa,as interpolated for 18?C from spinach data as determined byJordan and Ogren, 1984)     

Effects of Constant and Variable Nitrogen Supply on Sunflower (Helianthus annuus L.) Leaf Cell Number and Size

The effects of nitrogen (N) availability on cell number andcell size, and the contribution of these determinants to thefinal area of fully expanded leaves of sunflower (Helianthusannuus L.) were investigated in glasshouse experiments. Plantswere given a high (N =315 ppm) or low (N=21 ppm) N supply andwere transferred between N levels at different developmentalstages (5 to 60% of final size) of target leaves. The dynamicsof cell number in unemerged (< 0.01 m in length) leaves ofplants growing at high and low levels of N supply were alsofollowed. Maximum leaf area (LA_max) was strongly (up to two-fold)and significantly modified by N availability and the timingof transfer between N supplies, through effects on leaf expansionrate. Rate of cell production was significantly (P<0.05)reduced in unemerged target leaves under N stress, but therewas no evidence of a change in primordium size or in the durationof the leaf differentiation–emergence phase. In fullyexpanded leaves, number of cells per leaf (N_cell), leaf areaper cell (LA_cell) and cell area (A_cell) were significantly reducedby N stress. WhileLA_cell and A_cellresponded to changeover treatmentsirrespective of leaf size, significant (P<0.05) changes inN_cellonly occurred when the changeover occurred before the leafreached approx. 10% of LA_max. There were no differential effectsof N on numbers of epidermal vs. mesophyll cells. The resultsshow that the effects of N on leaf size are largely due to effectson cell production in the unemerged leaf and on both cell productionand expansion during the first phase of expansion of the emergedleaf. During the rest of the expansion period N mainly affectsthe expansion of existing cells. Cell area plasticity permitteda response to changes in N supply even at advanced stages ofleaf expansion. Increased cell expansion can compensate forlow N_cellif N stress is relieved early in the expansion of emergedleaves, but in later phases N_cellsets a limit to this response.Copyright 1999 Annals of Botany Company Helianthus annuus, leaf expansion, leaf cell number, leaf cell size, nitrogen, leaf growth, sunflower.     

Leaf Characteristics and Net Gas Exchange of Diploid and Autotetraploid Citrus

The effect of tetraploidy on leaf characteristics and net gasexchange was studied in diploid (2x ) and autotetraploid (4x) ‘Valencia’ sweet orange (Citrus sinensis (L.)Osb.) and ‘Femminello’ lemon (Citrus limon (L.)Burm. f.) leaves. Comparisons between ploidy levels were madeunder high irradiance (I) in a growth chamber or low total Iin a glasshouse. Tetraploids of both species had thicker leaves,larger mesophyll cell volume and lower light transmittance thandiploids regardless of growth I. Mesophyll surface area perunit leaf area of 2x leaves was 5–15% greater than on4x leaves. Leaf thickness and mesophyll cell volume were greaterin high I leaves than low I leaves. In high I, average leafarea was similar for 2x and 4x leaves, whereas in low I it was30% greater in 4x than in 2x leaves. Nitrogen and chlorophyllconcentration per cell increased with ploidy level in both growthconditions. The ratio of chlorophyll a:b was 25% greater in2x than in 4x leaves. When net CO₂assimilation rate (A_CO2) wasbased on leaf area, 4x orange leaves had 24–35% lowerA_CO2than their diploids. There were no significant differencesin A_CO2between 2x and 4x orange or lemon leaves when expressedon a per cell basis. Overall, lower A_CO2per unit leaf area oftetraploids was related to increase in leaf thickness, largermesophyll cell volume, the decrease in mesophyll area exposedto internal air spaces, and the lower ratio between cell surfaceto cell volume. Such changes probably increased the resistanceto CO₂diffusion to the site of carboyxlation in the chloroplasts. Cell volume; chlorophyll; irradiance; leaf thickness; nitrogen; photosynthesis; ploidy; Citrus limon ; C. sinensis ; ‘Valencia’ sweet orange; ‘Femminello’ lemon     

Hypothyroidism alters diaphragm muscle development

Sieck, Gary C., Louise E. Wilson, Bruce D. Johnson, andWen-Zhi Zhan. Hypothyroidism alters diaphragm muscle development. J. Appl. Physiol. 81(5):1965-1972, 1996.The impact of hypothyroidism (Hyp) onmyosin heavy chain (MHC) isoform expression, maximum specific force(P_o), fatigability, and maximumunloaded shortening velocity(V_o) wasdetermined in the rat diaphragm muscle (Dia) at 0, 7, 14, 21, and 28 days of age. Hyp was induced by treating pregnant rats with6-n-propyl-2-thiouracil (0.05% indrinking water) beginning at gestational day10 and was confirmed by reduced plasma levels of3,5,3-triiodothyronine and thyroxine. MHC isoforms wereseparated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by densitometry. IsometricP_o and fatigue resistance of theDia were measured in vitro at 26°C, andV_o was determined at 15°C with the slack test. Compared with control muscles,expression of MHC-slow was higher and expression of adult fast MHCisoforms was lower in Hyp Dia at all ages. The neonatal isoform of MHC continued to be expressed in the Hyp Dia until day28. At each age,P_o and fatigability were reducedand V_o was slowerin the Hyp Dia. We conclude that Hyp-induced alterations in MHC isoform expression do not fully predict the changes in Dia contractile properties.

     

Effects of soil resistance to root penetration on leaf expansion in wheat (Triticum aestivum L.): composition, number and size of epidermal cells in mature blades

Wheat seedlings {Triticum aestivum L.) were grown on soils withcontrasted resistances to root penetration (measured as penetrometerresistance, R_s. High R_s reduced the rates of leaf appearanceand expansion. Although the duration of expansion was increased,mature leaves were smaller. Underlying changes in leaf anatomywere investigated on cleared mature leaves, focusing on theepidermes. Three leaves were analysed: leaves 1 and 3 whichstarted their development in the embryo, and leaf 5 which wasinitiated on the seedling, after imposition of contrasted soilconditions. In all leaves, high R_s, caused a reduction in maturecell sizes, lengths and widths, and a shift in the relativeproportions of functionally different cell types, with a decreasein the relative proportions of stomata and associated cell types(interstomatal and sister cells) and an increase in the proportionsof unspecialized elongated epidermal cells and of trichomes.In leaves 3 and 5 the number of cellular files across the bladewas also reduced, while in leaf 1 it was similar at the twoR_s. These differences between leaves are attributed to differencesin their developmental stage when root stress was first perceived.Remarkably, R_s had no effect (leaf 1) or relatively small effects(leaves 3 and 5) on the total number of cells per file, suggestingthat this parameter is either largely insensitive to variationin root environment, or is programmed at the outset before stresswas perceived at the apex. Key words: Wheat, anatomy, mature epidermis, root impedance     

Chlorophyll Metabolism in Higher Plants VI. Involvement of Peroxidase in Chlorophyll Degradation

The following phenolics were found to be essential for peroxidase-dependentchlorophyll bleaching: 2,4-dichlorophenol (DCP), p-coumaricacid (HCA), phenol, p-hydroxyphenylacetic acid, p-hydroxybenzoicacid, p-hydroxyacetophenone, resorcinol and umbelliferone. Mostof them are monophenols with electron-attracting groups at thep-position. The short-lived radicals generated by horseradishperoxidase (HRP)-phenolics-H₂O₂ reaction might be involved inthis reaction. Tobacco leaf enzyme preparation with peroxidaseactivity for guaiacol could also degrade chlorophyll with suchphenolics. In addition, tobacco leaf methanol extract couldsubstitute for chlorophyll bleaching as an electron donor inthe absence of phenolics. In place of free H₂O₂, the glycolate-glycolateoxidase (GOX) system could degrade chlorophyll in [peroxidase$phenolics]-dependentbleaching. This chlorophyll bleaching system was inhibited by peroxidaseinhibitors, radical scavengers, reducing reagents, and carotenoids.Ascorbate and glutathione stopped chlorophyll bleaching withGSSG reductase and NADPH. The role of ascorbate and glutathionein peroxidase activity for controlling the chlorophyll degradationrate is discussed. (Received January 28, 1985; Accepted July 23, 1985)     

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm

Alterations of mitochondrial-encoded subunits of the F_oF₁-ATPsynthase are frequently associated with cytoplasmic male sterility(CMS) in plants; however, little is known about the relationshipof the nuclear encoded subunits of this enzyme with CMS. Inthe present study, the full cDNA of the gene TaF_Ad that encodesthe putative F_Ad subunit of the F_oF₁-ATP synthase was isolatedfrom the wheat (Triticum aestivum) fertility restorer ‘2114’for timopheevii cytoplasm-based CMS. The deduced 238 amino acidpolypeptide is highly similar to its counterparts in dicotsand other monocots but has low homology to its mammalian equivalents.TaF_Ad is a single copy gene in wheat and maps to the short armof the group 6 chromosomes. Transient expression of the TaF_Ad–GFPfusion in onion epidermal cells demonstrated TaF_Ad's mitochondriallocation. TaF_Ad was expressed abundantly in stem, leaf, anther,and ovary tissues of 2114. Nevertheless, its expression wasrepressed in anthers of CMS plants with timopheevii cytoplasm.Genic male sterility did not affect its expression in anthers.The expression of the nuclear gene encoding the 20 kDa subunitof F_o was down-regulated in a manner similar to TaF_Ad in theT-CMS anthers while that of genes encoding the 6 kDa subunitof F_o and the subunit of F₁ was unaffected. These observationsimplied that TaF_Ad is under mitochondrial retrograde regulationin the anthers of CMS plants with timopheevii cytoplasm. Key words: CMS, F_Ad subunit, F_oF₁-ATP synthase, retrograde regulation, wheat Received 8 October 2007; Revised 9 January 2008 Accepted 28 January 2008     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

Oxygenated cytochrome o (Vitreoscilla) formed by treating oxidized cytochrome with superoxide anion

Oxygenated cytochrome o can be formed experimentally in twoways, i) by reaction of reduced cytochrome o with molecularoxygen, or ii) by reaction of oxidized cytochrome o with superoxideanion generated by the action of the xanthine oxidase system.It is thermodynamically feasible for oxidized cytochrome o plusO₂–, and reduced cytochrome o plus O₂ to appear as intermediatesin reactions i) and ii), respectively. Superoxide dismutase completely inhibits the xanthine oxidase-catalyzedconversion of oxidized cytochrome o into the oxygenated formbut it has relatively little effect on the oxygenated cytochromeo formation in the reaction system consisting of NADH, NADH-cytochromeo reductase, and cytochrome o. Thus, if superoxide anion doesplay a significant role in the latter system it must be efficientlycoupled to react with cytochrome o and inaccessible to superoxidedismutase. Direct electron transfer from the reductase to thecytochrome without the involvement of superoxide anion is analternative mechanism. (Received December 16, 1976; )     

Regulation of Leaf Shape in the Solanifolia Mutant of Tomato (Lycopersicon esculentum) by Plant Growth Substances

The solanifolia mutant (sf/sf) of tomato (Lycopersicon esculentum)produces leaves consisting of leaflets with entire margins,unlike the lobed leaflets of normal plants. Normal plants treatedwith gibberellic acid (GA₃) produced leaves with entire marginswhereas mutant plants exposed to 2-chloroethyl-trimethyl ammoniumchloride (CCC)—an inhibitor of gibberellin biosynthesis—producedlobing of leaflets. The leaf area of the mutant was significantlygreater than that of the normal, but was not significantly differentfrom GA₃-treated normal leaves. Similarly, in CCC-treated mutantleaves the leaf area was not different from that of normal untreatedleaves. These observations suggest that the sf/sf mutation affectsthe leaf shape through its effect on endogenous gibberellinsand/or inhibitory substances. Leaf shape, Lycopersicon esculentum, plant growth substances, tomato