Effect of Phytochrome and Kinetin on Nitrite Reductase Activity in Zea mays

Red light and kinetin (10 µm) increased nitrite reductase(NIR) activity by 85 and 47% respectively in excised leavesof etiolated Zea mays. The stimulatory effect of kinetin decayedslower than that of red light. Indoleacetic acid (10 µm)had no effect on NIR activity. In the presence of abscisic acid(10 µm), the kinetin stimulated increase in NIR activitywas totally nullified, however, the red light irradiated plantsretained 20–25% increase in NIR activity over the darkcontrol. If ABA was given 2 h after kinetin treatment or redlight irradiation, it totally blocked kinetin stimulation asnoticed earlier, but red light stimulation was inhibited byonly 11%. Kinetin-treated and the red light irradiated leavesshowed 20–25% increase in nitrate accumulation, whichwas totally nullified by ABA. The experiments presented suggestan independent mode of signal transduction by kinetin and phytochromein stimulating NIR activity. (Received December 2, 1986; Accepted February 7, 1987)     

Photoactivation of NADP-Malate Dehydrogenase by a Heterogeneous Photochemical System in Zea mays

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction in order to activate the NADP-malate dehydrogenase in light. The photocatalyst acridine orange or proflavin sensitized EDTA-dependent reduction of ferredoxin. In a complete system, consisting of a dye donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified NADP-MDH was observed in vitro. The chloroplast mediated redox activation of enzyme essentially required ferredoxin, while heterogeneous photochemical mediated activation of enzyme need not require ferredoxin. The heterogeneus photochemical system activated NADP-MDH by eight fold similar to chloroplasts mediated ferredoxin dependent redox activation but was not affected by the presence of disalicylinden propanediamine-1, 2-disulphonic acid while there was complete inhibition of chloroplasts mediated activation of NADP-MDH in presence of this inhibitor. These observations suggest that a thiol mediator is essential for reductive activation of NADP-MDH and ferredoxin is not required for photochemical activation.     

Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the K(m) value for phosphoenolpyruvate is 80mum. When assayed at 30 degrees C, the enzyme shows normal Michaelis-Menten kinetics, but when assayed at 45 degrees C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by P(i), by SO(4) (2-) and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.     

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.     

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.     

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.     

Interrelationships between Potassium, Calcium, and Magnesium Nutrition of Zea mays L.

The uptake of potassium, calcium, and magnesium ions by maizeand the interrelationships among the cations have been investigatedat 48 K: Ca: Mg ratios in culture solutions. Calcium was foundto stimulate K⁺ and Mg⁺⁺ uptake at certain cation ratios butinhibit it at others. Potassium did the same for Ca⁺⁺ uptake,and Mg⁺⁺ for Ca⁺⁺ and K⁺. The uptake of Mg⁺⁺ was generally enhancedby K⁺. The sum of the cations in the plants expressed in meqwas fairly constant for treatments of the same K⁺ concentrationat the low to moderate levels of K⁺, but at considerably higher(> 24 meq l^–1) K⁺ levels the constancy was not dependenton K⁺ concentration. Potassium depressed, but Mg⁺⁺ stimulatedphosphorus accumulation. Calcium stimulated phosphate absorptionat certain cation ratios but had no effect at others. The plantyield increased with increasing K⁺ up to 24 meq l^–1 ofK⁺ after which the yield tended to fall with further increasein K⁺. The yield was also increased by Ca⁺⁺. Magnesium increasedthe yield at certain cation ratios and either depressed it orwas without effect at others.     

Ribonuclease Activity Associated With Ribosomes of Zea mays

At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles.

The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered.

The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured.

     

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.     

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.     

Respiration and Mitochondrial Activity in Zea mays Roots As Affected by Osmotic Stress

Studies were made of metabolism in highly vacuolated and slightlyvacuolated Zea mays root tissue both during and after plasmolysis. Plasmolysis resulted in decreased respiration and carbon dioxideevolution from glucose and an increased sucrose synthesis. Inhibitionof respiration during plasmolysis in both the highly vacuolatedand slightly vacuolated tissue was not relieved by supply ofglucose, organic acids, or uncouplers of oxidative phosphorylation.Mitochondria isolated from plasmolysed tissue were tightly coupled,but activity in vitro was inhibited by exposure to a high negativeosmotic potential. It is suggested that low TCA cycle activityin vivo must be due either to inhibition of mitochondrial activityor to reduced flow of carbon through the glycolytic pathway. A low potential for TCA cycle activity after deplasmolysis issuggested, as addition of pyruvate stimulated carbon dioxideevolution but not oxygen uptake, which was severely decreased.This was presumably due to severe mitochondrial damage as shownby their activity in vitro. However, it is not clear whetherrespiration in vivo is rate limited by rapid leakage of metabolicintermediate (reported earlier) or by lysis of mitochondria. Deplasmolysis did not damage mitochondria from slightly vacuolatedtissue, a result which was consistent with respiratory measurementsmade in vivo. The data show that mitochondria in vacuolated tissue are damagedduring and after deplasmolysis and not before. It is suggestedthat lysis of mitochondria occurs in vivo as a result of a sharpincrease in the osmotic potential of the cell fluids.     

Dehydrogenase and urease activities of maize (Zea mays L.) field soils

Summary Dehydrogenase and urease activities, bacterial and fungal populations and physicochemical characteristics of maize (Zea mays L.) field soils have been studied for one crop cycle. A comparison has been made among soils of three different agricultural systemsviz permanent agriculture on plain lands in valleys, recently introduced terrace land agriculture and age old ‘slash and burn’ type of shifting agriculture on slopes. Results demonstrate that the enzyme activities, microbial population as well as most of the physico-chemical characteristics of soils followed the trend permanent agriculture on plain lands>terrace land agriculture>‘slash and burn’ type of shifting agriculture. Moisture and nutrient levels and topography of the lands were found to be major factors responsible for the trend.     

Regulation of Mg,K-ATPase Activity in Guard Cells of Commelina communis by Phytochrome and ABA

The microsomal fraction obtained from guard cell protoplastswas assayed for ATPase activity at pH 6.5. Triton X-100 didnot affect this stimulation of activity of ATPase by K⁺ up to5 mM, but the detergent abolished the stimulatory effect ofK⁺ at higher concentrations. The ATPase activity was inhibitedby N,N-dicyclohexylcarbodiimide and ABA. Irradiation with redlight enhanced the ATPase activity more than did irradiationwith far-red light. ABA and irradiation with red or far-redlight were effective only in the presence of K⁺. These resultssuggest the possibility that the ATPase activity is modulatedonly indirectly by light and ABA. (Received January 9, 1989; Accepted July 10, 1989)     

Regulation of glutathione S-transferases of Zea mays in plants and cell cultures

An antiserum to glutathione S-transferase (EC 2.5.1.18) from maize (Zea mays L.) responsible for herbicide detoxification has been raised in rabbit. The antiserum was specific to the M_r 26000 subunit of the enzyme from maize seedlings and suspension-cultured cells, and recognized the isoenzymes active toward both atrazine and metolachlor. When plants were treated for 24 h with the herbicide antidote N,N-diallyl-2-2-dich-loroacetamide (DDCA), enzyme activities toward metolachlor were doubled in the roots and this was associated with a 70% increase in immunodetectable protein. Translation of polysomal RNA in vitro showed that the increase in the transferase in root tissue was brought about by a ninefold increase in mRNA activity encoding the enzyme. Treatment of suspension-cultured cells with cinnamic acid, metolachlor and DDCA raised enzyme activities but did not increase synthesis of glutathione S-transferase. In cultured maize cells, enzyme synthesis was maximal in mid-logarithmic phase, coinciding with the highest levels of enzyme activity. When callus cultures were established from the shoots of a maize line known to conjugate chloro-s-triazines, enzyme activity towards atrazine was lost during primary dedifferentiation. However, levels of total immunodetectable enzyme and activity toward metolachlor were increased in cultured cells compared with the parent shoot tissue.     

Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria

L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780-784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K_m for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD⁺-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments.     

Activation of ATPase Activity in the Chromatin Fraction of Pea Nuclei by Calcium and Calmodulin

ATPase, especially the Ca^2$-dependent enzyme, in the chromatinfraction isolated from nuclei of pea seedlings was activatedby bovine brain calmodulin and by protein that was judged tobe calmodulin from its Ca^2$-dependent activation of NAD kinase.This calmodulin-dependent ATPase activity was blocked by calmodulin-specificinhibitors. (Received July 11, 1983; Accepted October 24, 1983)     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.