Ethylene Release from Leaves of Xanthium strumarium L. and Zea mays L.

The release of ethylene into sealed Erlenmeyer flasks by intactleaves and leaf discs of Xanthium strumarium L. a C₃ plant andZea mays L. a C₄ plant were compared both in white light andin darkness. The effects of the presence or absence of addedCO₂ (in the form of sodium bicarbonate) the photosynthetic inhibitor3-[3,4-dichlorophenyl]-l, l-dimethyl urea (DCMU) and 1-aminocyclopropane-1-carboxylicacid (ACC), the precursor of ethylene in higher plants, werealso investigated. The rate of ethylene release from leaf tissue of Xanthium inthe absence of added CO₂ was markedly reduced in the light (i.e.at the CO₂ compensation point). Treatments that would enhancethe CO₂ availability to the tissue (i.e. added bicarbonate,darkness, treatment with DCMU) allowed higher levels of ethylenerelease. Incubation of the tissue with ACC considerably enhancedthe release of ethylene compared to that from the correspondingcontrol tissue without ACC. However, the pattern of ethylenerelease induced by the various treatments was similar with orwithout added ACC. When tissue, in the absence of added CO₂, was transferred fromlight to darkness, and back to light for 90 min periods, theethylene release rates Increased during the interposed darkperiod but resumed the lower rate during the final light period.The addition of CO₂ in the light resulted in a similar rateof ethylene release to that found in the dark. The overall pattern of ethylene release from Zea leaf tissuesubjected to light and dark in the presence or absence of addedCO₂ was similar to that of Xanthium. However, two or three timesmore ethylene was released from maize leaves in the light whenCO² was added compared to that generated in the dark. This isin marked contrast to Xanthium, where, under the light conditionsused, the ethylene release rate in the dark equalled or exceededthat occurring in the light, even in the presence of high levelsof CO₂. A very low rate of ethylene release was observed atthe CO₂ compensation point of maize. A speculative model is presented to explain how photosyntheticactivity might act as a key factor in regulating ethylene evolutionfrom leaf tissue in these experiments. It invokes the conceptof an inhibition by CO₂ of ethylene retention or breakdown thuspermitting more ethylene to be released from the leaves.     

Auxin and Ethylene Control of Growth in Seedlings of Zea mays L. and Avena sativa L

The effects of applied ethylene on the growth of coleoptilesand mesocotyls of etiolated monocot seedlings (oat and maize)have been compared with those on the epicotyl of a dicot seedling(the etiolated pea). Significant inhibition of elongation by ethylene (10 µll^–1for 24 h) was found in intact seedlings of all three species,but lateral expansion growth was observed only in the pea internodeand oat mesocotyl tissue. The sensitivity of the growth of seedlingparts to ethylene is in the decreasing order pea internode,oat coleoptile and oat mesocotyl, with maize exhibiting theleast growth response. Although excised segments of mesocotyland coleoptile or pea internode all exhibit enhanced elongationgrowth in IAA solutions (10^–6–2 ? 10^–5 moll^–1), no consistent effects were found in ethylene. Ethyleneproduction in segments was significantly enhanced by applicationof auxin (IAA, 10^–5 mol l^–6 or less) in all tissuesexcept those of the eat mesocotyl. Segments of maize show a slow rate of metabolism of applied[2-¹⁴C]IAA (30 per cent converted to other metabolites within9 h) and a high capacity for polar auxin transport. Ethylene(10 µl l^–1 for 24 h) has little effect on eitherof these processes. The oat has a smaller capacity for polartransport than maize and the rate ef metabolism of auxin isas fast as in the pea (90 per cent metabolized in 6 h). Althoughethylene pretreatment does not change the rate of auxin metabolismin oat, there is a marked reduction in auxin transport. It is proposed that the insensitivity of maize seedlings toethylene is related to the supply and persistence of auxin whichcould protect the seedling against the effects of applied orendogenously produced ethylene. Although the mesocotyl of oatis sensitive to applied ethylene it may be in part protectedagainst ethylene in vivo by the absence of an auxin-enhancedethylene production system. The results are discussed in relationto a model for the auxin and ethylene control of cell growthin the pea.     

Regulation of Nitrate Reductase Activity in Corn (Zea mays L.) Seedlings by Endogenous Metabolites

Primary and secondary metabolites of inorganic nitrogen metabolism were evaluated as inhibitors of nitrate reductase (EC 1.6.6.1) induction in green leaf tissue of corn seedlings. Nitrite, nitropropionic acid, ammonium ions, and amino acids were not effective as inhibitors of nitrate reductase activity or synthesis. Increasing α-amino nitrogen and protein content of intact corn seedlings by culture techniques significantly enhanced rather than decreased the potential for induction of nitrate reductase activity in excised seedlings.     

Control of Nitrate Uptake by Phloem-Translocated Glutamine in Zea mays L. Seedlings

Abstract: The putative role of glutamine, exported from leaves to roots, as a negative feedback signal for nitrate uptake was investigated in Zea mays L. seedlings. Glutamine (Gln) was supplied by immersion of the tip-cut leaves in a concentrated solution. Nitrate (NO₃⁻) uptake was measured by its depletion in amino acid-free medium. The treatment with Gln resulted in a strong inhibition of nitrate uptake rate, accompanied by a significant enrichment of amino compounds in root tissue. The effect of N-availability on NO₃⁻ uptake was determined in split-root cultures. The plants were subjected to complete or localized N supply. Inducible NO₃⁻ uptake systems were also induced in N-deprived roots when the opposite side of the root system was supplied with KNO₃. The inhibitory effect of Gln was unaffected by localized N supply on one side of the split-root. The potential role of Gln in the shoot-to-root control of NO₃⁻ uptake is discussed.     

Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: I. Regulation by Nitrate Flux

The roles that leaf nitrate content and nitrate flux play in regulating the levels of nitrate reductase activity (NRA) were investigated in 8- to 14-day old maize (Zea mays L.) plants containing high nitrate levels while other environmental and endogenous factors were constant. The nitrate flux of intact plants was measured from the product of the transpiration rate and the concentration of nitrate in the xylem. NRA decreased when the seedlings were deprived of nitrate. The nitrate flux and the leaf nitrate content also decreased. When nitrate was resupplied to the roots, all three parameters increased.     

Impaired Anthocyanin Production in Barbiturate treated Zea mays Seedlings

Anthocyanin production was impaired in both roots and shootsof Zea mays seedlings germinated on 1 and 2 mM concentrationsof four barbiturates having different lipid/aqueous partitioncoefficients. The severity of impaired anthocyanin productionwas greater in those seedlings treated with the higher lipidsoluble barbiturates irrespective of the concentrations used.Indirect evidence is presented which indicates that barbituratesinterfere with normal membrane physiology responsible for anthocyaninproduction. Anthocyanin, barbiturate, seedlings, Zea mays     

Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea mays L. Leaves

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10(-5)m and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions.     

Etioplast Development in Dark-grown Leaves of Zea mays L

The ultrastructure of etioplasts and the acyl lipid and the fatty acid composition of sequential 2-centimeter sections cut from the base (youngest) to the top (oldest) of nonilluminated 5-day-old etiolated leaves of Zea mays L., and the acyl lipid and fatty acid composition of the etioplasts isolated from them have been investigated. There is a 2.5-fold increase in the size of the plastids from the base to the tip of the leaf, and an increase both in the size of the prolamellar body and in the length of lamellae attached to it. The etioplasts in the bundle sheath and mesophyll cells of the older, but not the younger leaf tissue, are morphologically distinct. The monogalactosyl and digalactosyldiglycerides, phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were the only detectable acyl lipids in the isolated etioplast fractions. Together with phosphatidylethanolamine these were also the major acyl lipids in the whole leaf sections. With increasing age of the leaf tissue, increases occurred in two of the major plastid lipids, monogalactosyldiglyceride and phosphatidylglycerol, while the levels of essentially nonplastid lipids remained constant or declined slightly. The monogalactosyldiglyceride to digalactosyldiglyceride ratio increased from 0.4 to 1.1 in the tissue sections of increasing age and from 0.7 to 1.2 in the etioplasts isolated from them. Similarly, the galactolipid to phospholipid ratio increased from 0.8 to 1.4 in the tissue and from 0.5 to 4.5 in the isolated plastids. In the latter, the proportions of phosphatidylglycerol (as a per cent of total phospholipid) increased from 20 to 41% with increasing age of plastids.

Linolenic acid was the major fatty acid in the total lipid of each of the etioplast fractions, but it was only the major fatty acid in the total lipid of the oldest leaf tissue. Its proportion in both total lipid extracts and individual lipids increased with age. The trans Δ³ hexadecenoic acid was absent from all lipids. The protochlorophyllide content of the tissue increased with age. The results are discussed in relation to the use of illuminated etiolated leaves for studying chloroplast development.

     

Regulation of Assimilatory Sulfate Reduction by Cadmium in Zea mays L

Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from ³⁵SO₄²⁻ into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5′-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5′-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves.     

Cytosolic Ascorbate Peroxidase in Seedlings and Leaves of Maize (Zea mays)

Ascorbate peroxidase (APX) was purified to homogeneity frommaize (Zea mays L. cv.) coleoptiles. APX was a monomer witha molecular mass of 28 kDa, as determined by gel nitration andSDS-polyacrylamide gel electrophoresis. It contained one protohememoiety per molecule, with the oxidized form giving a Soret peakat 403 nm with small peaks at 502 and 638 nm, and the reducedform giving peaks at 435 and 556 nm. The enzyme was not inactivatedby depletion of ascorbate. Cell fractionation and immunohistochemicalstudies using polyclonal antibodies raised against maize APXrevealed that the enzyme was not located in the chloroplastsof green leaves. It was abundant in the cytoplasm but not inthe vacuoles of cells in the coleoptile, mesocotyl and youngleaves of seedlings. In mature green leaves, small amounts ofthe enzyme were distributed in vascular systems, in particularin the companion cells. The N-terminal amino acid sequence ofmaize APX exhibited high homology to pea cytosolic APX, spinachAPX and Arabidopsis APX, but not to APX from tea chloroplasts. (Received February 15, 1993; Accepted May 6, 1993)     

Cyanide-resistant Respiration and Cold Resistance in Seedlings of Maize (Zea mays L.

Respiratory activity of mitochondria isolated from seedlingsof two cold-resistant and two cold-susceptible maize cultivarswas examined. The greater potential for cyanide-resistant respirationfound in the cold-resistant seedlings raises the possibilityof a role for the alternative respiratory pathway in cold-resistance. alternative respiration, cold-resistance, cyanide-resistant respiration, maize, mitochondria, Zea mays     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.     

Regulation of IBA synthetase from maize (Zea mays L.) by drought stress and ABA

The rate of indole-3-butyric acid (IBA) synthesis in maize seedlingsis dependent on the culture conditions of the plants. When theseedlings were grown on filter paper soaked with different amountsof water, the activity of IBA synthetase differed strongly.High amounts of water (150 and 200 ml per bowl) inhibited IBAsynthesis completely in vitro, whereas 30 and 50 ml water perbowl increased the activity dramatically. Under conditions whereIBA synthetase was inhibited (150 ml H₂O), an increase of enzymeactivity was observed when abscisic acid (ABA) was exogenouslyadded in concentrations between 510^–4 to 510^–7M. Under ‘drought’ conditions (50 ml H₂O per bowl)the same ABA concentrations were inhibitory. Jasmonic acid andsalicylic acid also enhanced IBA synthetase activity to someextent, whereas indole-3-acetic acid (IAA) and kinetin had noeffect. Activity could also be enhanced by osmotic stress (NaCIand sorbitol), but not under temperature stress. In accompanyinginvestigations the endogenous contents of IAA, IBA, and ABAunder the different culture conditions have been determinedas well as the energy charge of the seedlings. Similar observationshave been made with Amaranthus, wheat and pea seedlings Key words: Abscisic acid, Amaranthus paniculatus, drought stress, inole-3-butyric acid biosynthesis, Pisum sativum, Triticum aestivum, Zea mays     

Age, Fluence Rate, and Anthocyanin Production in Zea mays Seedlings

Increase in fluence rates of white light over the range of 5to 80 µmol m^–2 s^–1 brought about a correspondingincrease in amounts of anthocyanin production in shoots of Zeamays L. seedlings. Roots also exhibited a similar relationshipbetween increased fluence rate and increased anthocyanin productionover the range of 5 to 40 µmol m^–2 s^–1 whereasfluence rates above 40 µmol m^–2 s^–1 broughtabout decreases in anthocyanin production. Rates of productionand amounts of accumulation of anthocyanin in both shoots androots were found to vary with the age of the seedlings at thetime of exposure to light. Age, fluence rates, anthocyanin, seedlings, Zea mays     

Purification and Characterization of Two Benzoyl-l-Tyrosine p-Nitroanilide Hydrolases from Etiolated Leaves of Zea mays L

Two benzoyl-l-tyrosine p-nitroanilide hydrolases (BTPAases I and II) were purified from the etiolated leaves of Zea mays L. and characterized. BTPAase I was electrophoretically homogeneous and consisted of two identical subunits having a molecular weight of 53,000. The molecular weight of BTPAase II was 65,000. The Michaelis constants for substrate, BTPA, were 4 millimolar and 1.3 millimolar for BTPAases I and II, respectively. Based on the action of various inhibitors on both enzyme activities, these enzymes were classified as serine proteases. BTPAase I showed caseinolytic activity at neutral pH and the activity was strongly inhibited by the serine protease inhibitors.     

Limitations to the Measurement of In Vitro Nitrate Assimilation by Exogenous Additives and Endogenous Interference Factors in the Leaves of Zea mays L. Seedlings

An evaluation of existing assay procedures for the measurementof nitrate assimilation in the leaves of Zea mays L. has highlightedlimitations in established in vitro assay techniques. Both exogenouslyadded compounds and endogenous leaf components affected theresults of an in vitro nitrate reductase (NADH: oxido-reductase,EC 1.6.6.1 [EC] .) assay. Reducing agents employed as enzyme protectantswere excluded from the assay in order to accurately measurethe concentration of nitrogen compounds by colorimetric andHPLC analysis. Endogenous nitrate levels in a leaf extract asmeasured by these two analytical techniques indicated significantinterference in the colorimetric method due to the presenceof various organic compounds. This interference was most apparentat low nitrate concentrations, however, changes in nitrate concentrationappeared to be more closely comparable between the two techniques.In addition, endogenous leaf components also interfered withthe precise determination of nitrite that had accumulated duringan in vitro nitrate reductase assay. These endogenous factorsacted directly upon the colorimetric assay of nitrite by a concentration-dependentreaction with the diazotizing reagent sulphanilamide. The interferingcomponents were of low molecular weight ( 5000 daltons) andeasily separable from nitrate reductase by molecular sieve chromatography.Their interference in the nitrite assay could only be partiallyprevented by heating or storage, while other treatments studied,including those frequently used to terminate an in vitro assaysuch as zinc acetate precipitation or chloroform extraction,had less effect in alleviating the interference. Similar endogenouscomponents which affected the colorimetric assay of nitritewere also found in leaf extracts from wheat, pea, soybean andsunflower seedlings. Zea mays L., nitrate reductase, reducing agents, plant interference factors     

Effects of Salinity on Water Transport of Excised Maize (Zea mays L.) Roots

The root pressure probe was used to determine the effects of salinity on the hydraulic properties of primary roots of maize (Zea mays L. cv Halamish). Maize seedlings were grown in nutrient solutions modified by additions of NaCl and/or extra CaCl₂ so that the seedlings received one of four treatments: Control, plus 100 millimolar NaCl, plus 10 millimolar CaCl₂, plus 100 millimolar NaCl plus 10 millimolar CaCl₂. The hydraulic conductivities (Lp_r) of primary root segments were determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. Exosmotic hydrostatic Lp_r for the different treatments were 2.8, 1.7, 2.8, and 3.4·10⁻⁷ meters per second per megapascals and the endosmotic hydrostatic Lp_r were 2.4, 1.5, 2.7, and 2.3·10⁻⁷ meters per second per megapascals, respectively. Exosmotic Lp_r of the osmotic experiments were 0.55, 0.38, 0.68, and 0.60·10⁻⁷ meters per second per megapascals and the endosmotic Lp_r were 0.53, 0.21, 0.56, and 0.54·10⁻⁷ meters per second per megapascals, respectively. The osmotic Lp_r was significantly smaller (4-5 times) than hydrostatic Lp_r. However, both hydrostatic and osmotic Lp_r experiments showed that salinization of the growth media at regular (0.5 millimolar) calcium levels decreased the Lp_r significantly (30-60%). Addition of extra calcium (10 millimolar) to the salinized media caused ameliorative effects on Lp_r. The low Lp_r values may partially explain the reduction in root growth rates caused by salinity. High calcium levels in the salinized media increased the relative availability of water needed for growth. The mean reflection coefficients of the roots using NaCl were between 0.64 and 0.73 and were not significantly different for the different treatments. The mean values of the root permeability coefficients to NaCl of the different treatments were between 2.2 and 3.5·10⁻⁹ meters per second and were significantly different only in one of four treatments. Cutting the roots successively from the tip and measuring the changes in the hydraulic resistance of the root as well as staining of root cross-sections obtained at various distances from the root tip revealed that salinized roots had mature xylem elements closer to the tip (5-10 millimeters) compared with the controls (30 millimeters). Our results demonstrate that salinity has adverse effects on water transport and that extra calcium can, in part, compensate for these effects.     

Lateral Root Anlage Development in Excised Roots of Vicia faba L., Pisum sativum L., Zea mays L. and Phaseolus vulgaris L.

The development of lateral root primordia has been investigatedin excised roots of Vicia faba, Pisum sativum, Zea mays andPhaseolus vulgaris cultured in White's medium supplemented with2 per cent sucrose and compared with previously published dataon such development in primaries of the corresponding intactplants (control roots). Primordia were produced in each batchof excised roots over the 6 day culture period but at a lowerrate (number day^–1) than in the controls. Such primordia in cultured roots of Zea and Phaseolus completedtheir development and grew out as lateral roots over a periodsimilar in length to that found in the controls, but with acell number of only about 33 per cent of that attained at thetime of secondary emergence in the primaries of the latter roots.These lower cell numbers were at least partly a reflection ofincreases in mean cell doubling time over the period of anlagedevelopment investigated in the excised roots relative to thecorresponding values found in the controls. Primordia initiated in excised roots of Pisum and Vicia didnot complete their development in culture, i.e. no lateral rootsemerged and arrest took place with cell numbers of only 37 (Pisum)and 17 (Vicia) per cent of the numbers determined at the timeof secondary root emergence in the controls. Such arrested primordiahad few nuclei in S and none in mitosis. Moreover, at leastin Pisum, the frequency distribution of the relative DNA contentof the nuclei in the latter primordia approximated that foundin the apical meristem of primary roots following the establishmentof the stationary phase under conditions of carbohydrate starvation. It has also been demonstrated in the course of these investigationsthat lateral root primordium development in all four speciesis at least biphasic and possibly triphasic. Vicia faba L., broad bean, Pisum sativum L., garden pea, Zea mays L., maize, Phaseolus vulgaris L., dwarf bean, root primordia, anlage, cell doubling time, lateral root emergence