Maize Scutellum Research Articles

Purification and characterization of three non-allelic alcohol dehydrogenase isozymes in Maize

Three non-allelic alcohol dehydrogenase (alcohol:NAD + oxidoreductase, EC 1.1.1.1) isozymes have been purified to homogeneity from maize scutellum. The three isozymes, numbered 1, 2 and 3, have similar subunit molecular weights ( M r 35 000) as judged by SDS-polyacrylamide gel electrophoresis, and similar holoenzyme molecular weights ( M r 70 000) as judged by gel filtration chromatography and sedimentation equilibrium centrifugation. Each isozyme exhibits a pH optimum at pH 10 although they are not stable at this pH. Isozyme 1 is more thermolabile than isozyme 2 or isozyme 3. All three isozymes share similar substrate specificities and Michaelis constants. However, isozyme 1 can better utilize primary alcohols with longer carbon chains and has a lower Michaelis constant for acetaldehyde.

Kinetic properties of acid phosphatase from scutella of germinating maize seeds

Acid phosphatase purified from maize scutellum showed a kinetic transition from negative cooperativity at pH 5.4, to Michaelian behavior at pH 6.7. The

Expression of genetically distinct superoxide dismutases in the maize seedling during development

AbstractImmunoassays for the cytosolic and mitochondrial superoxide dismutases (SOD) of maize were developed and used to study the expression of these proteins in the maize seedling. The genetically distinct proteins, SOD‐3 and SOD‐4, are preferentially expressed in the scutellum, comprising approximately 1% of the total water‐soluble protein of that tissue. SOD‐2, SOD‐3, and SOD‐4 are synthesized in the scutellum during early sporophytic development, probably on cytosolic ribosomes. Two‐dimensional gel electrophoresis of crude scutellar extracts indicates that significant changes occur in the protein composition of the maize scutellum following seed imbibition. Using the immunoassays, a maize line exhibiting a significant reduction in cyanide‐sensitive SOD protein was identified.

Cytoplasmic pH of maize scutellum cells

The cytoplasmic pH of scutellum cells from 3-day-old maize seedlings was estimated from the distribution of 2, 4-dinitrophenol (DNP) between scutellum

Regulation of Glyoxysomal Enzyme Expression in Maize

The activity levels of three glyoxysomal enzymes (catalase, isocitric lyase, and malate synthase) were measured in the scutellum following germination of the inbred lines W64A, R6-67, and A16. In W64A, as in most maize lines examined, germination was accompanied by a rapid and synchronous increase in the activities of all three enzymes, and reached a peak at about day 4 and declined thereafter. In R6-67, catalase activity continues to increase past day 4 and reaches its highest activity level on later days. In A16, catalase activity is very low due to the lack of expression of the Cat2 gene. Despite these significant differences in catalase expression, the levels of the other two glyoxysomal enzymes did not differ in these inbred lines. Artificial inhibition of catalase in W64A by exogenous application of 10–4 M aminotriazole did not inhibit germination, nor did it alter the levels of the other two glyoxysomal enzymes. Similarly, application of 10–4 M itaconate to W64A seeds inhibited the appearance of isocitric lyase, but did not inhibit germination or alter the levels of malate synthase or catalase. Comparative cell fractionation and immunological studies were conducted with W64A and A16 and their microbodies were observed under the electron microscope. Cell fractionation studies were also conducted with W64A seeds germinated in the presence of aminotriazole or itaconate. Thus, our results suggest that the expression of these three glyoxysomal enzymes is not regulated coordinately in the maize scutellum.

Acid phosphatase from maize scutellum: Succinylation and some kinetic properties of the active enzyme

Acid phosphatase purified from maize scutellum did not dissociate into subunits upon acylation with succinic anhydride. The enzyme maintained its catalytic activity after succinylation of 52 free amino groups permolecule. The results also showed that free amino groups may play an important role in the maintenance of enzyme stability at pH values greater than 5.4.

Sucrose—proton efflux from maize scutellum cells

Sucrose—proton efflux was studied using maize scutellum slices treated with mannose, an energy poison in maize scutellum cells. High rates of sucrose

Properties of acid phosphatase from scutella of germinating maize seeds

One acid phosphatase (optimum pH at 5.4) was purified from maize scutellum after 96 hr of germination. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulfate (SDS). The enzyme has a MW of 65 000 ± 4000 as determined by Sephadex G-200 gel filtration and SDS-PAGE. The enzyme contained 16% neutral sugars, and cations are not required for activity. The purified enzyme was not inactivated by DTNB at pH 8. The hydrolysis of glucose-6-phosphate in the presence of 4 mM fluoride and 4 mm EDTA, at pH 6.7 (optimum pH), seems to be catalysed by this acid phosphatase.

Biochemical Characterization of a Catalase Inhibitor from Maize

Some biochemical properties of the catalase inhibitor purified from maize scutella are described. The inhibitor is heat-labile and its activity is destroyed by trypsin, indicating that it is a protein. It does not appear to be a lectin nor does the inhibition involve proteolysis. The active inhibitor is a dimer with each subunit having a molecular weight of 5600 as determined by sodium dodecyl sulfate electrophoresis. A kinetic analysis performed in the presence of increasing levels of inhibitor gave unusual Lineweaver-Burk patterns. Possible explanations for these patterns are discussed. The inhibitor is active against all catalases tested from a wide variety of organisms.

Sucrose Efflux from the Maize Scutellum

AbstractSucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb+ uptake took place during sucrose efflux. In mannose, rates of Rb+ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose‐H+ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.

Genetic regulation of the catalase developmental program in maize scutellum: Identification of a temporal regulatory gene

Genetic and biochemical analyses suggest that the developmental program of catalase (H(2)O(2):H(2)O(2) oxidoreductase, EC 1.11.1.6) activity in maize scutella is controlled by a temporal regulatory gene (Car1) that is distinct from the structural genes thus far identified. Recombination data show that Car1 is located about 37 map units from the Cat2 structural gene on the chromosome 1S. Turnover studies indicate that Car1 may act by regulating the rate of catalase synthesis.

Glycosylation of catalase inhibitor necessary for activity

A protein isolated from maize scutella which inhibits catalase in vitro has been shown to contain 12% carbohydrate in the form of galactose. This corresponds to four galactose molecules per inhibitor subunit. Removal of the carbohydrate with β-galactosidase or blockage with a galactose-specific lectin abolished activity of the inhibitor.

Invertase and maltase in the free space of the maize scutellum

When maize scutellum slices were incubated in solutions of sucrose or maltose, there was a release of glucose into the bathing solution. The pH optima for glucose release were 2.5 for sucrose and 3.5 for maltose. From measurement of rates of glucose uptake into slices in the presence or absence of sucrose, it is calculated that glucose uptake will introduce errors of 3–9%, depending on the sucrose concentration, in estimates of free-space sucrose-hydrolase activity at pH 2.5. At their respective pH optima, maltose was hydrolysed at a rate 2.5 times that of sucrose. When frozen-thawed slices were used the same pH optima were obtained, but rates of hydrolysis were increased. Raffinose and melezitose also were hydrolysed with pH optima of 2.5 and 3.5, respectively. α-Methyl glucose was not hydrolysed. A 60-min HCl treatment (pH 2) of scutellum slices destroyed 69% of the sucrose-hydrolase activity and 100% of the maltose-hydrolase activity. In contrast, sucrose uptake and sucrose synthesis from exogenous fructose were not affected by HCl treatment. It is concluded that there are two hydrolases, acid invertase and maltase; that they are either on or outside the plasmalemma (in the free space); and that they are not necessary to the disaccharide uptake processes either by supplying exogenous hexose or by acting as transporters.

Developmental Expression of Genetically Defined Peptidases in Maize

The activities of genetically defined amino- and endopeptidases of maize were compared in pericarp, endosperm, and embryonic tissue of the maize kernel from 5 days postpollination until harvest. Activities were highest in the immature stages and declined as drying progressed. The expression of some of the peptidase genes contributed by the pollen parent was examined during early endosperm development in an F(1) cross. The paternally contributed peptidase variants could first be detected 7 days after pollination.During germination and early seedling growth the peptidase activities continued to decline in the endosperm but maintained high levels in the scutellum. The maize peptidases probably have no direct role in hydrolysis of endosperm storage proteins during germination and growth.The peptidase isozymes were associated with the soluble cytoplasmic fraction of the maize scutellum during early growth. Only one tissue specificity was found for the peptidases. Occurrence of a particular aminopeptidase isozyme, AMP2, is limited to endosperm tissue during kernel maturation.

Evaluation of a catalase inhibitor in the developmental regulation of maize catalase

AbstractA protein which has been shown to inhibit catalase in vitro appears to vary inversely with catalase activity in the maize scutellum during early sporophytic development when assayed using a catalase inhibition assay. This result suggested that the inhibitor protein may play a direct role in regulating catalase activity during this time period.Four experimental approaches were used to evaluate this putative regulatory role, including immunological quantitation of individual catalase isozymes during germination using rocket immunoelectrophoresis, perturbation of normal catalase expression with hydrogen peroxide or allylisopropylacetamide (AIA), examination of a mutant line with an altered catalase developmental program, and direct radioimmunoassay of the inhibitor protein during germination. The results of these experiments indicate that the quantitative changes in catalase activity during development are not mainly due to changes in the expression of the catalase inhibitor. Other possible roles of this protein in catalase regulation are discussed.

Properties of a plasmalemma ATPase of the maize scutellum

Properties of a plasmalemma phosphatase of the maize scutellum, tentatively identified as an ATPase in a previous paper, were investigated. Fresh and frozen-thawed scutellum slices, that had been treated with 10 mM HCl to destroy acid phosphatases, were used as a source of enzyme. With the exceptions of the Na +, K + and dinitrophenol experiments, the two kinds of slices gave similar results. ATP and CTP were the best substrates for the enzyme followed by TTP, UTP, CDP, ADP and GTP. UDP, nucleoside monophosphates, sugar phosphates, inorganic pyrophosphate and p-nitrophenyl phosphate were relatively ineffective as substrates. The K m's for ATP and ADP were 0.65 and 5 mM, respectively, but the two substrates gave the same V max (49.8 μmol P i/hr/g slices). Previously, it was shown that the products of ATP hydrolysis are ADP, AMP and P i. Using these previous results and from the time courses of ATP disappearance from the bathing solution and the appearance of P i and ADP, it was concluded that ATP and ADP were hydrolysed by the same enzyme. The ATPase was not inhibited by oligomycin. N- N′-Dicyclohexylcarbodiimide (DCCD) was a poor inhibitor, and a water soluble analog of DCCD, 1-ethyl-3 (3 dimethyl-aminopropyl)-carbodiimide, gave only 33% inhibition. The relative effectiveness of divalent cations for stimulating ATPase activity was Mn 2+ > Mg 2+ ⩾ Ca 2+ > Co 2+ · Na + and K + gave a small additional stimulation in the presence of Mg 2+. However, Na + and K + gave a much greater stimulation when no divalent cation was added, and this occurred only when fresh slices were used. Dinitrophenol also increased ATPase activity only when fresh slices were used. Since it is likely that both the uptake of Na + and K + and the action of dinitrophenol would lower the electrochemical gradient of protons across the plasmalemma, the different results obtained with fresh slices indicate that the ATPase in these slices was under the constraint of a proton gradient.

Localization of a phosphatase (ATPase) on the plasmalemma of the maize scutellum

A phosphatase (ATPase) was demonstrated on the surface of the maize scutellum cell by showing that (1) when exogenous ATP was hydrolysed by intact scutellum cells, ADP, AMP and P i appeared in the bathing solution in stoichiometric amounts, (2) the rate of hydrolysis was sensitive to bathing solution pH; (3) exogenous Mg 2+ increased the rate of hydrolysis and (4) when the ATPase reaction was carried out in the presence of lead nitrate, TEM photographs showed lead phosphate deposits located almost exclusively in the plasmalemma. The ATPase was tightly bound to the plasmalemma and was not destroyed by freezing and thawing scutellum slices, a treatment which disrupted the plasmalemma. Acid treatment (10 mM HCl) of fresh or frozen-thawed scutellum slices destroyed acid phosphatase activity but had little effect on ATPase activity at pH 6.5. Following acid treatment of the scutellum slice preparations, a definite Mg 2+ requirement for ATPase activity could be demonstrated.

Nitrate Reductase and Soluble Cytochrome c Reductase(s) in Higher Plants

The 4S cytochrome c (Cyt c) reductase activity of several plant species was markedly stimulated by cyanide and ferrocyanide but those of the 8S nitrate reductase component and other particulate components of the maize (Zea mays L.) scutellum by comparison, were increased only slightly. The effect of cyanide and ferrocyanide was not due to elimination of cytochrome oxidase interference but resulted from the stimulation of NADH-dependent reduction of Cyt c. A 4S Cyt c reductase component which could be isolated by ammonium sulfate fractionation and diethyl-aminoethyl-cellulose chromatography was found to be stimulated markedly by cyanide and ferrocyanide. The remaining 4S Cyt c reductase, which was insensitive to cyanide and ferrocyanide, was also fractionated with ammonium sulfate into two components. One of these, like the 8S Cyt c reductase, was sensitive to a protease from the maize roots which is relatively specific for nitrate reductase. This 4S Cyt c reductase species could be a subunit of nitrate reductase.

A model for sucrose transport in the maize scutellum

A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N-ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.

Identification of embryonal antigens of maize: Globulins as primary reserve proteins of the embryo

Normal and γ-irradiated caryopses of Zea mays L. were germinated, and the degradation of embryonal antigens (EA) was followed in the endosperms, scutella and embryonic axes of the seedlings, using double immunodiffusion, immunoelectrophoresis and quantitative immunoprecipitation. The predominant transient EA were presumed to be storage proteins related to the reserve globulins of dicotyledonous seeds. Therefore globulins were isolated from maize scutella, purified by (NH4)2SO4 fractionation and isoelectric precipitation, and the molecular weights of the polypeptide units were estimated by discontinuous sodium-dodecyl-sulphate slab electrophoresis. The globulins were found to be identical with the predominant EA and amounted to about 40% of the protein nitrogen in the embryos of mature, non-germinated caryopses. The presumed reserve function of the globulins and the characteristic time course of their degradation in embryonic axes and scutella of maize seedlings are discussed in relation to the two-step pattern of mobilization of nitrogen reserves in germinating cereal caryopses.