Dimethipin (2,3-Dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4-tetraoxide) Effects on Soybean Seedling Growth and Metabolism

Three-day-old dark-grown soybean [Glycine max (L.) Merr.] seedlingswere transferred to 2 mM CaSO₄ or 10^–5 M dimethipin in2 nM CaSO₄ and root-fed via liquid culture. Plants were placedin continuous darkness or in continuous white light (200 µE.m^–2?s^–11,PAR) at 25?C. Dimethipin inhibited root and shoot elongationin dark-grown plants after 24 h and 48 h, respectively. In thelight, root elongation was inhibited also after 24 h, but hypocotylelongation was not significantly affected. Extractable phenylalanineammonia-lyase (PAL) activity per axis in dimethipin-treateddark-grown axes was not generally affected but, in the lightdimethipin caused a significant decrease in PAL activity (24to 96 h). Total soluble hydroxyphenolics in axes were not affectedby dimethipin in light- or dark-grown plants. Anthocyanin andchlorophyll levels were lowered in hypocotyls of dimethipin-treatedplants after 48 to 96 h. Soluble protein in hypocotyls of light-or dark-grown seedlings was not substantially affected by dimethipin.Nitrate reductase (NR) activity (per organ) was generally notaffected by dimethipin in light-grown cotyledons, but in theroots of these seedlings, NR activity was significantly decreased.Proteolytic enzyme activity using three substrates (leucine-p-nitroanilide,LPNA; proline-p-nitroanilide, PPNA; and benzoylarginine-p-nitroanilide,BAPA) indicated little effect on enzyme activities per organin roots and hypocotyls. These data suggest that dimethipinat low concentrations can cause significant growth inhibitionin soybean seedlings grown in either light or darkness and thatfurthermore, extractable activities of some enzymes associatedwith nitrogen metabolism and secondary metabolism are alteredby this chemical. Light also plays a role in the activity ofthis chemical. (Received November 29, 1983; Accepted January 25, 1984)     

Distribution of nitrate reductase in ageing bean seedlings

The in vivo activity of nitrate reductase (NR, E.C. 1.6.6.1 [EC] )in the roots, stem and leaves of bean (Phaseolus vulgaris L.)was measured at different ages of seedlings. The leaves alwayshad higher levels of the enzyme than the roots or stem. Thelevel of the enzyme in the very young leaves were low, increasingto a maximum by day 10 to 11 of seedling growth at 26°C,after which it start to decline. The level of the enzyme in7 dayold decotyledonized leaves was about 2.5 times higher thanthat in leaves from intact seedlings. A supply of exogenousnitrate caused a considerable increase in the total organicnitrogen in the leaf only after day 9, when the nitrogen supplyfrom the cotyledons presumably is low. (Received March 22, 1975; )     

Triadimenol Increases Nitrate Levels and Nitrate Reductase Activity in Canola Leaves

Nitrate reductase activity in the first true leaves of canola(Brassica napus L.) seedlings grown in one-quarter strengthHoagland's solution from seeds pretreated with triadimenol (0.3or 30 g (a.i.) kg^–1 of seed) was higher than controlsduring the growth period of 15 to 25 d after planting. Triadimenolalso increased chlorophyll levels, the increase being more pronouncedat its lower concentration. The treatment also increased theweight and nitrate content of the leaves. When seedlings weregrown in nutrient solution containing 1 to 20 mM nitrate, theincrease in nitrate reductase activity by triadimenol was higherat lower rather than at higher nitrate concentrations. The nitratelevels and Kjeldahl nitrogen in the triadimenol-treated leaveswas higher than the controls at concentrations of added nitrateabove 2 mM. Addition of nitrate to plants grown in ammonium,increased nitrate reductase activity more in plants grown fromtriadimenol-treated seeds than controls. However, addition of10µM triadimenol for 24 h to ammonium-grown plants hadlittle effect on enzyme activity, both in the absence as wellas the presence of nitrate. This study demonstrates that triadimenolincreases nitrate reductase activity and nitrate accumulationin the leaves and at least part of the increased enzyme activityis independent of nitrate accumulation. Key words: Triazoles, nitrate content, nitrate reductase activity     

Induction of nitrate reductase in pumpkin seedlings

Nitrate reductase activity (NRA) was found to be induced in9-day-old pumpkin seedlings by nitrate and light. NRA was greatestin leaves and cotyledons and in vitro measurements gave highervalues than in vitro measurements. NRA was found in roots bythe in vivo method but not by the in vitro method. NRA changedwith the age of the seedling with maximum activity in 7-day-oldcotyledons and 9-day-old roots of light grown plants; and rootsof 7-day-old etiolated plants. Little activity was found inetiolated cotyledons. ¹ Present address: College of Agriculture and Animal Husbandry,P.O. Box 32, Rezaiyeh, Iran (Received September 30, 1977; )     

Biosynthesis of Ferredoxin-Nitrite Reductase in Rice Seedlings

Changes in ferredoxin-nitrite reductase [EC 1.7.7.1 [EC] ] in etiolatedrice seedlings were followed during induction by nitrate andlight. Etiolated seedlings showed maximal induction of the enzymeactivity during greening with nitrate, while the enzyme activityin etiolated seedlings receiving nitrate in darkness increasedhalf as much as that in nitrate-treated greening plants. Theincrease in nitrite reductase activity during induction coincidedwith an increase in the content of proteins immunoprecipitatedby antibodies raised against spinach nitrite reductase. Lighthad no effect on the induction of the extractable nitrite reductasein the absence of nitrate. Poly(A)⁺-RNA extracted from nitrate-treatedgreening shoots directed the synthesis in a rabbit reticulocyte-lysateof polypeptides immunoprecipitated by spinach nitrite reductaseantibodies. One major polypeptide larger than the native enzymewas found among the translation products, suggesting that nitritereductases in greening rice shoots are synthesized as an precursorform. Analysis of two-dimensional electrophoretograms indicatedthe existence of isoforms of nitrite reductase in rice seedlingswhich had been immunoprecipitated with spinach nitrite reductaseantibodies. ¹To whom all correspondence should be sent. (Received May 15, 1987; Accepted September 7, 1987)     

The Relationship of Abscisic Acid to Nitrate Reductase Activity in the Potato Plant

Abscisic acid (ABA) at 3.8 µM suppressed both in vivoand in vitro nitrate reductase activity in roots, stems andleaves of potato plants grown in solution culture. Suppressionwas maximal between 24 and 48 h, followed by recovery of activityat 72 h in roots and leaves and at 96 h in stems. Removal from ABA after 24 h resulted in complete recovery ofnitrate reductase activity in roots by 24 h and partial recoveryin leaves. ABA treatment enhanced nitrate accumulation in roots,decreased that of leaves, but had no effect on stem nitratecontent. ABA enhanced decay of the enzyme following nitrate removal;by 7 h activity in roots was 22.5% of the initial value comparedto 55% in the control. ABA showed a less drastic effect on lossof activity in leaves and stems. These results indicate thatABA suppression of nitrate reductase activity is not dependenton nitrate uptake, and although it reduced leaf nitrate contentthere was no clear relationship between tissue nitrate levelsand the ABA response. (Received September 13, 1984; Accepted July 1, 1985)     

Inverse Correlation of Thiol Proteinase with Nitrate Reductase Activities in Barley Leaves

Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml^–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml^–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)     

The Effect of Abscisic Acid on Amino Nitrogen and Protein Content of Potato Plants in Relation to the Inhibition of Nitrate Reductase Activity

Treatment of potato plants grown in nutrient solution with 3.8µM ABA resulted in reduced soluble protein in roots andin leaves at 24 h, but not in stems. This treatment reducedin vivo nitrate reductase activity in all organs for about 48h with the most pronounced reduction occurring in the roots.Excised root and leaf segments from plants treated with ABAfor 24, 48 and 72 h absorbed significantly more ¹⁴C leucine,compared to the control but the percent incorporation into proteinwas not altered in roots. In response to ABA total free amino nitrogen in leaves was lowerat 5 and 72 h and in stems at 72 h. Amino nitrogen content ofroots was enhanced by ABA at 5, 24 and 72 h due to generallyhigher levels of aspartate, serine, glutamate, proline and ammonia.There was no consistent relationship between ABA suppressionof nitrate reductase activity and ammonia or specific aminoacid (except proline) levels in leaves and stems. The increasedfree amino nitrogen levels in response to the hormone may bethe result of impaired NO₃– reduction rather than thecause. The results of protein synthesis studies and solubleprotein content suggest that ABA inhibition of nitrate reductaseis not due to general inhibition of protein synthesis and mayinvolve specific inhibition of nitrate reductase protein synthesis. ¹ Contribution No. 684, Department of Plant Science, Universityof Manitoba.     

Variation in Nitrate Reductase Activity in Lolium

Nitrate reductase activity was studied in the leaves and rootsof Lolium perenne. Growth temperatures of 8, 15, or 20 °Cdid not affect activity, but the same temperatures during assayhad differential effects on the nitroso couple used to measureenzyme activity. Activity increased with increasing light intensity,reaching a high plateau value at around 40 W m^–2. Nitratecontent of leaves, also measured in this experiment, did notvary significantly with different light intensities. Increasingnitrate in the nutrient solution up to 0.5 mM N also increasedactivity. Adding ammonium chloride at similar levels to thenitrate caused no marked repression of activity. Removal ofnitrate from the nutrient solution decreased enzyme activitywithin 24 h. Marked diurnal fluctuations occurred in activity,apparently in response to light intensity, since the nitratelevel in the plant varied little. The enzyme activity of rootswas much less than that of leaves. In the parents and progeny from a half diallel cross, the parentalgenotypes differed significantly in activity, but the numberof families involved was too small for the regression of progenyon parents (b = 1.74) and the correlation coefficient (r = 0.44NS) to achieve significance. In this experiment a significantpositive regression was obtained between nitrate reductase activityand dry matter yield.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Partitioning and redistribution of sulphur during S-stress in Macroptilium atropurpureum cv. Siratro

During the first 7 d of sulphate-deprivation stored SO₄^2- wasredistributed and assimilated into organic forms in the tropicallegume Macroptilium atropurpu-reum cv. Siratro. However, whilstthe sulphate content of all tissues declined after removingthe external SO₄^2- supply this was slowest in mature leaves.By contrast, the total S content of mature leaves declined markedlyin the absence of external sulphate whilst that of both youngleaves and roots increased. Furthermore, when radiolabelledSO₄^2- was applied to abraded surfaces of mature leaves, mostof the translocated label was recovered in the root following2 d SO₄^2- deprivation. By contrast, radiolabelled SO₄^2-appliedto young leaves was mostly retained in these tissues and nottranslocated. Within 3 d of removing the SO₄^2- supply there was a large increasein extractable APS-sulphotransferase activity in roots accompaniedby a decline in nitrate reductase activity, but these effectswere not seen in leaves. Five days after the removal of SO₄^2-there was a large increase in the content of asparagine in roots. The results are discussed in relation to the co-ordination ofNO₃^- and SO₄^2- uptake and assimilation and the partitioningof sulphur during S-stress. Key words: Sulphate supply, stomatal conductance, ATP-sulphurylase, APS-sulphotransferase, nitrate reductase     

Effects of Nitrate Withdrawal and Resupply on the Assimilation of Nitrate in Leaves of Tomato

Nitrate reduction in leaves of tomato occurred at the same ratein plants grown in 8.0 mol m^–3 nitrate as in plants grownin 2.0 mol m^–3 nitrate, but at a much slower rate in plantsgrown in 0.1 mol m^–3 nitrate. However, the plants grownin 8.0 mol m^–3 nitrate had a larger leaf system than theplants grown in 2.0 mol m^–3 nitrate, and so the totalcapacity to assimilate nitrate was greater in the plants grownin the higher concentration. It was shown that plants grownin 8.0 mol m^–3 nitrate were better buffered against nitratewithdrawal than plants grown in 2.0 mol m^–3 nitrate asthe rate of nitrate reduction declined more slowly when plantswere transferred to 0.1 mol m^–3 nitrate from the higherconcentration than from the lower concentration. Furthermore,leaf expansion continued in the plants transferred from thehigher concentration, whereas it ceased abruptly in the plantstransferred from the lower concentration. It was concluded thatboth continuing expansion and continuing nitrate reduction wereaccompanied, and possibly caused by, a release of nitrate fromstorage pools in the lower part of the stem or the roots. Duringwithdrawal of nitrate the leaves were shown to maintain potentialactivity of the enzyme nitrate reductase although there wasno nitrate to be reduced. When nitrate was resupplied it couldbe reduced very quickly and reduction in the leaves was seento increase within 5 h of resupply. By 3 d after resupply furtherenzyme activity had been induced. Key words: Lycopersicon esculentum Mill, nitrate assimilation, nitrate reductase activity, nitrate withdrawal     

Nitrate Reductase and Nitrite Reductase Activity in Dark-Grown Radish Seedlings: Relation to the Supply of NADPH

Functioning of nitrate reductase and nitrite reductase was measured in intact cotyledons from radish seedlings (Raphanus sativus L.) grown in the dark in a nitrate medium. Reduction of nitrate to nitrate did proceed during the whole period of 45 h, whereas the reduction of nitrite in the intact cotyledons dropped abruptly between 20 and 23 h after exposing the roots to nitrate. The activity of the enzymes glucose-6-P dehydrogenase (G6PDH) and 6-P-gluconate dehydrogenase (6PGDH), measured in cotyledon extracts, showed a sharp decline simultaneously with the drop in nitrite reductase activity of the intact cotyledons. It was concluded that the amount of NADPH generated by the enzymes G6PDH and 6PGDH is not sufficient to allow continuous functioning of nitrite reductase after 20 h in cotyledons of seedlings grown in the dark. Therefore, the results from our experiments point to the functioning of nitrite reductase as the rate limiting step in the reduction pathway of nitrate in the dark.     

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.     

Flavine nucleotide nitrate reductase from broad bean leaves

Hydrosulfite-reduced FMN served as an electron donor for nitratereductase purified from broad bean leaves. FMN was successfullyreplaced with BV. The flavine nucleotide nitrate reductase hadits pH optima at about 7.8 with phosphate buffer and at about7.4 with Tris-HCl buffer. The Km's for nitrate and FMN were3.7 ? 10^–4 M and 3.7 ? 10^–5 M, respectively. NADH₂: nitrate reductase activity was completely inhibited by0.1 mM p-CMB, whereas FMNH₂: nitrate reductase activity wasnot. Inhibited activity was restored by the addition of cysteine.A sulfhydryl enzyme is involved in the NADH₂: nitrate reductasesystem but not in the FMNH₂ : nitrate reductase system. NADH₂and FMNH₂ probably feed electrons into the electron transportchain at different sites. The nitrate reductase preparationhad an NADH₂-specific diaphorase activity which was almost completelyinhibited by 0.1 mM p-CMB. The NADH₂-specific diaphorase mayform the sulfhydryl enzyme which mediates electron transferbetween NADH₂ and nitrate. (Received May 6, 1969; )     

Influence of Ethephon on Nitrate Reductase, Protein Amino Nitrogen and Nitrate Content of Solanum tuberosum L.

Nitrate reductase activity was stimulated in roots and stems,but suppressed in leaves of potato plants grown in nutrientculture by 30 mg.liter^–1 ethephon applied to the culturesolution. In stems, nitrate reductase activity was stimulatedafter 5 h and by 24 h it was more than two fold that of thecontrol. The magnitude of stimulation by ethephon was less inroots compared to stems. Ethephon treatment enhanced ethyleneproduction by roots, stems and leaves but the level of productionwas not significantly different in these organs. The stimulationof nitrate reductase activity was prevented by cycloheximideand cordycepin suggesting the involvement of new protein synthesis. Ethephon enhanced TCA precipitable protein levels in both rootsand stems while that in leaves was not significantly affected.Amino nitrogen content increased in parallel with protein contentin response to ethephon, with roots exhibiting substantial stimulation.Nitrate accumulation in stem tissues was not affected by ethephontreatment but was increased in roots at 24 and 48 h. Leaf NO³content declined with time in both ethephon-treated and controlplants and after 24 h significantly less NO³ accumulated intreated leaves. These results are explained in terms of ethephonstimulated protein synthesis and increase in cellular metabolismand permeability. (Received August 21, 1984; Accepted January 7, 1985)     

O-Benzylhydroxylamine: An Inhibitor of Phenylpropanoid Metabolism in Plants

O-Benzylhydroxylamine (OBHA) is a potent inhibitor of phenylalanineammonialyase (PAL, EC 4.3.1.5 [EC] ) and phenylpropanoid metabolismas evidenced by its effects on three plant species [soybean(Glycine max (L.) Merr.), buckwheat (Fagopyrum esculentum Moench.),and mung bean (Vigna radiata L.)]. When supplied to roots, OBHA(10^–5 M) did not significantly inhibit light- or dark-growthof soybean seedlings, but reduced (25%) soluble hydroxyphenoliccompound accumulation in light-grown axes. Higher concentrations(510^–5 M) of OBHA caused reductions (25%) in axis freshweight of light-grown seedlings (72 h), but did not lower axisweight of dark-grown seedlings. Anthocyanin accumulation inhypocotyls of intact mung bean seedlings was reduced by 25%after 3 days light growth after treatment with OBHA (10^–5M) via root feeding. Anthocyanin content of excised, etiolatedbuckwheat hypocotyls floated on solutions of OBHA (10^–5M) and incubated in the light for 24 h was reduced by 40%. L-Phenylalanineand t-cinnamic acid, intermediates of phenylpropanoid metabolism,were able to partially reverse this inhibition in buckwheat.Extractable PAL activity (specific activity basis) in soybeanaxes was substantially reduced (20% in dark, 40% in light) asearly as 24 h after root feeding with OBHA (10^–5 M). Reductionof PAL activity (specific activity or per axis basis) by OBHAcompared to control levels, continued throughout a time courseof 96 h. Kinetic studies on soybean PAL revealed a K_m of 1.1mM for L-phenylalanine and an apparent K_i of 3.5 µM forOBHA. (Received May 31, 1985; Accepted August 6, 1985)     

Variation in the Content and Composition of Sterols in Alfalfa Seedlings Treated with Compactin (ML-236B) and Mevalonic Acid

Compactin (ML-236B), a specific inhibitor of 3-hydroxy-3-methylglutarylCoA reductase, inhibited the elongation of roots and hypocotylsof Medicago sativa seedlings when it was applied to the roots.Addition of mevalonic acid, the direct product of the enzyme,together with compactin relieved the growth inhibition of roots. The contents and compositions of sterols were studied in threeparts of M. sativa seedlings—roots, hypocotyls and cotyledons.Compactin (20 µM) decreased the sterol contents of rootsand hypocotyls by about a half but did not affect that of cotyledons.On the other hand, mevalonic acid (2 mM) increased the sterolcontent of roots more than threefold the nontreated controllevel but not the contents of hypocotyls and cotyledons. Mevalonicacid added in combination with compactin had a similar effecton the sterol content of roots as when it was added alone. The major sterol in all three parts was stigmasterol whetheror not compactin or mevalonic acid was present. However, thevariation of the sterol composition in the roots was distinct;mevalonic acid-treated roots markedly accumulated ⁷-sitosterol,24-methylenecycloartanol and squalene. (Received October 16, 1986; Accepted April 2, 1987)