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.

     

Protein Changes in Response to Pyrene Stress in Maize （Zea mays L.） Leaves

Phytoremedlation is a relatively new approach to remove polycyclic aromatic hydrocarbons （PAHs） from the environment. When plants are grown under pyrene treatment, they respond by synthesizing a set of protective proteins. To learn more about protein changes in response to pyrene treatment, we extracted total proteins from the leaves of maize （Zea mays L.） 1 week after pyrene treatment. The proteins extracted were separated with twodimensional gel electrophoresis. In total, approximately 54 protein spots were found by comparing gels from treated and control groups. According to the Isoelectric point, molecular weight, and abundance of these protein spots, 20 pyrene-lnduced proteins were found to have changed abundance. Of these, 15 protein spots were Increased and five protein spots were newly appeared in pyrene-treated plant leaves. Six model upregulated protein spots of different molecular weights were excised from the gels and subjected to trypsin digestion followed by peptide separation using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptlde masses were used to search the matrix-science database for protein Identification. Two of the proteins were Identified on the basis of the homology of their peptide profiles with existing protein sequences as pyruvate orthophosphate diklnase and the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunlt. These proteins are Involved in the regulation of carbohydrate and energy metabolism. The present study gives new Insights into the pyrene stress response In maize leaves and demonstrates the power of the proteomlc approach in phytoremedlation of PAHs.     

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays

An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-¹⁴C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 × 10⁻⁸ molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-¹⁴C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin.     

[3H]Indole-3-Acetyl-myo-Inositol Hydrolysis by Extracts of Zea mays L. Vegetative Tissue

[³H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37°C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as α-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other fraction enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.     

The Hydrolysis of Endosperm Protein in Zea mays

Degradation of the major storage proteins in maize endosperm, zein and glutelin, begins during the 2nd day of germination. The protein most abundant in the mature endosperm is degraded most rapidly. The patterns of protein loss are essentially similar in germinating seeds and excised endosperms. Cycloheximide, added at the beginning of the incubation period, prevents the development of α-amylase and protease activities and the disappearance of starch and protein reserves. Late additions (70 hours) of cycloheximide still inhibit the increase in hydrolase activity but have no effect on the hydrolysis of storage reserves. The results indicate that the hydrolytic enzymes are synthesized de novo in the maize endosperm.     

Stomatal Responses to Pressure Changes and Interruptions in the Water Supply of Detached Leaves of Zea mays L

Stomata of Zea mays L. respond to changes in hydrostatic pressure in the water supply of the leaves almost instantaneously and in all leaf parts simultaneously. Therefore, the leaf is a hydraulic unit. The stomata are part of it and their aperture is controlled by the water potential in the water-conducting system. Stomatal aperture is not uniquely related to the relative water content of a leaf. The relation depends also on the humidity in the air and is different for the upper and the lower epidermis.     

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)     

Water Deficit and Inflorescence Development in Zea mays L.

A water deficit imposed during the period of terminal male inflorescenceinitiation and early development reduced both the growth rateand the mature size of that organ in Zea mays (cv. Iochief).Growth and development of the axillary shoots, the potentialfemale inflorescences, was inhibited during the episode of waterdeficit but promoted thereafter. As a result, plants which hadbeen subjected to a water deficit at that period produced 2–3mature cobs and relatively large axillary shoots at the lowernodes, whereas plants supplied with water throughout produceda single mature cob and relatively small axillary shoots. A water deficit imposed during other growth phases did not producethis response and, moreover, a further period of deficit imposedlater in development, following a deficit at the sensitive stage,inhibited the enlargement of the axillary shoots invoked bythe earlier deficit. It did not, however, inhibit the enhancedfloral development of those axillary shoots nor reverse theinhibition of tassel growth. The data are discussed in relation to correlative inhibitionin Zea mays.     

Longitudinal Water Movement in the Primary Root of Zea mays

The rates of transfer of tritiated water (THO) along lengthsof excised primary roots of Zea mays have been measured undera variety of conditions. The following values of ‘apparentdiffusion coefficients’ for THO in the root tissue havebeen evaluated: 1.5±0.1x10^-5 cm² sec^-1 in roots boiledfor 3 min before use,0.5±0.03x10^-5 cm² sec^-1 in rootspoisoned with 10^-2 M NaF,0.9±0.07x10^-5 cm² sec^-1 in rootspoisoned with 10^-2 M NaN₃,and 2.1±0.2x10^-5 cm² sec^-1in normal roots. The bathing medium in all cases was 1.0 mMKCl/0.1 mM CaCl₂ with the addition of the inhibitors where appropriate.Thefourfold increase in the rate of THO transfer in normal rootscompared with poisoned ones is attributed to the existence ofa long-distance convective flow in the first case, which isterminated by the addition of inhibitors. Since experimentsshow that this convective flow must occur both acropetally andbasipetally with equal velocity, it is thought to occur in thephloem.By assuming the ‘streaming transcellular strands’model for phloem transport, the rate of movement required togive the observed transfer has been computed as approximately4.5x10^-2 cm sec^-1 (160 cm h^-1).The earlier report of the existenceof a highly impermeable barrier surrounding the xylem vesselshas been further substantiated by the experiments reported here.     

Growth and Deposition of Inorganic Nutrient Elements in Developing Leaves of Zea mays L

Spatial distributions of growth and of the concentration of some inorganic nutrient elements were analyzed in developing leaves of maize (Zea mays L.). Growth was analyzed by pinprick experiments with numerical analysis to characterize fields of velocity and relative elemental elongation rate. Inductively coupled plasma and atomic emission spectroscopy were used to measure nutrients extracted from segments of leaf tissue collected by position. Leaves 7 and 8, both elongating 3 millimeters per hour had maximum relative elemental growth rates of 0.06 to 0.08 millimeters per hour with maximum rates 20 to 50 millimeters from the node and cessation of growth by 90 millimeters from the node. Spatial distribution of dry weight density revealed that the rate of biomass deposition was maximum in the most rapidly expanding region and continued beyond the elongation zone. The nutrient elements K, Cl, Ca, Mg, and P showed different distribution patterns of ion density (on a dry weight basis). K and Cl had minimal density in the leaf tips; K density was maximum in the growing region, whereas Cl density was maximum at the region of growth cessation. Ca, Mg, and P had relatively high densities at the base of the elongation zone near the node and also in the tip regions. Near the node, P and Mg densities were higher in the young, growing leaves, whereas Ca density near the node was higher in older leaves that had completed elongation. Deposition rates of all nutrients were greatest in the region of maximum elongation rate.     

Lipid Composition of Zea mays Seedlings and Water Stress-Induced Changes

The imposition of a polyethylene glycol-induced osmotic stressof –1.5 MPa for 48 h on 28 d old Zea mays (cv. Style Pak)seedlings resulted in a 44% decreased stem dry weight and increasedtriglyceride levels in stem and leaf tissues; increased sterylester levels also occurred in stems. The magnitude of theseincreases was such that, on a dry weight basis, there were increasedtotal triglyceride and steryl ester levels in the seedlingsafter the 48 h applied stress. In stems the increased triglyceridelevel was evident in all of the component fatty acids examined,whereas in leaves it was associated mainly with one fatty acidcomponent, viz. linolenic acid (C18: 3). Changes in sterol levels were small but significant and largelyrestricted to the stem. Proline levels of all three tissuesincreased in response to water-stressing the seedlings and thegreatest increase also occurred in the stem tissues.     

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

Distribution of [14C] Dichlorophenoxyacetic Acid in Cultured Zygotic Embryos of Zea mays L.

The uptake of 2,4-dichlorophenoxyacetic acid (2,4-D), necessary for the in vitro induction of callus formation and somatic embryogenesis in cultured immature maize embryos, was quantified after culture on nutrient medium with [¹⁴C]2,4-D. The identity of the ¹⁴C label in the embryos was determined by high performance liquid chromatography (HPLC), and its distribution within embryos was visualized on sections of plastic embedded material. Quantification of the ¹⁴C label after a pulse label of 16 h showed a hundredfold accumulation of 2,4-D in the embryos with respect to the initial medium concentration. During tissue processing for in situ detection of ¹⁴C, however, up to 70% of the label disappeared because of the embedding process. The best structural preservation was obtained after ethanol-mediated infiltration of Technovit 7100. Water-mediated infiltration of Technovit 7100 gave the highest retention of ¹⁴C. HPLC analysis showed that more than 95% of the residual ¹⁴C label found in embryos was still 2,4-D. Autoradiography showed that the embryogenic inbred line A188 contained ¹⁴C label in distinct regions of the scutellum, coleoptile, and suspensor. The nonembryogenic inbred line A632 contained more label after 16 h of culture in a different distribution compared with A188. Subculture of the embryos for 24 and 72 h and histologic analysis showed that cell proliferation and callus formation were restricted to specific regions of the embryo in both inbred lines. The pattern of 2,4-D distribution did not codistribute with regions of proliferation, indicating that 2,4-D is not the only trigger for proliferation. Received August 18, 1997; accepted February 12, 1998     

Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L

Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity.     

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.     

Metabolism of [14C]Indole-3-Acetic Acid by Coleoptiles of Zea mays L.

Reverse-phase high-performance liquid chromatography was usedto analyse [¹⁴C]-labelled metabolites of indole-3-acetic acid(IAA) in coleoptile segments of Zeo mays seedlings. After incubationfor 2 h in 10^–2 mol m^–3 [2-¹⁴C]IAA, methanolic extractsof coleoptiles contained between six and ten radioactive compounds,one of which co-chromatographed with IAA. The metabolic productsin coleoptile extracts appeared to be similar to those in rootextracts, with an oxindole-3-acetic-acid-like component as theprincipal metabolite, but the rate of metabolism was slowerin coleoptile than in root segments. Decarboxylation did notappear to play a major role in the metabolism of exogenous IAAduring the short incubation periods. Moreover, external IAAconcentration had little effect on the pattern of metabolism.Coleoptile segments were also supplied with [¹⁴C]IAA from agardonor blocks placed at the apical ends, and agar receiver blockswere placed at the basal ends. After incubation for 4 h, theidentity of the single radioactive compound in the receiverblocks was shown to be IAA by both reverse-phase high-performanceliquid chromatography and gas chromatography-mass spectrometrytechniques. Key words: Zea mays, Coleoptile, High-performance liquid chromatography, Indole-3-acetic acid     

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.