Rapid Decrease in the Glutamine Synthetase Activity during Imbibition of Thermodormant New York Lettuce Seeds

A high glutamine synthetase (GS) activity was detected in dryseeds of New York lettuce but it decreased rapidly during imbibitionat 35°C. The decrease in GS activity was remarkable at 35°C,but not so at 45°C or at 25°C. The activity of extractedGS was relatively constant even at 35°C. The decrease inGS activity stopped immediately after the addition of cycloheximide(CH). This suggests the presence of a GSinactivating systemin the seeds. The amount of ammonia increased abruptly duringthe early stage of imbibition at 35°C, suggesting the blockageof ammonia-assimilation at high temperature. The GS activitythat decreased to a low level during imbibition at 35°Cfor 12 h increased again during the subsequent imbibition atlow temperature (15CC) before the break of thermodormancy. Ahigher GS activity was found in the embryonic axes than in thecotyledons. Partial purification of GS showed that lettuce seedGS was eluted as a single peak on Biogel A1.5m or DEAE-Sephacel(mol wt: 4.4 x 10⁵). Thus the thermodormancy of New York lettuce seeds may be relatedto inactivation of GS during imbibition at high temperatures,and the activity of GS in the embryonic axes may control thegermination of New York lettuce seeds through the regulationof glutamine formation. (Received May 11, 1983; Accepted September 13, 1983)     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

Promotion by gibberellin of lettuce seed germiation as a function of presoaking period

Germination of lettuce seeds (Lactuca sativa L. cv. Grand Rapids)was examined in the presence of various doses (10^–5.0–10^–3.0M) of gibberellic acid applied at various times (hour 0–8)of soaking. Germination promotion by gibberellic acid was greateras the dose of gibberellic acid was increased and attenuatedwith the length of the presoaking period. As an exception, ca.95% germination was always evoked by the largest dose (10^–3M) of gibberellic acid given at any time of soaking. The dose-responsecurve obtained for each presoaking period had a distinct sigmoidalprofile. Synergistic and photoreversible promotion by red light of thegibberellin-induced germination was also investigated. Far-redlight pulse given 6 hr after the red pulse was still effectivein removing the red light action. Application of enzyme kineticsto the gibberellin action and also to the synergism betweengibberellin and red light was suggested. ¹National Institute for Basic Biology, Myodaiji, Okazaki 444,Japan. (Received December 18, 1979; )     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)     

The appearance of glutamine synthetase in turions of Spirodela polyrhiza (L.) Schleiden as regulated by blueand red light, nitrate and ammonium

Control by light and nitrogen (nitrate and ammonium) of theappearance of glutamine synthetase (GS; EC 6.3.1.2 [EC] ) in turionsof Spirodela polyrhiza (L.) Schleiden, strain SJ, was investigatedduring the pregermination period, i.e. up to 48 h after onsetof light. Immediately after transfer from after-ripening conditions(5C, darkness, D) to germination conditions (25C), GS activitydid not respond to light or nitrate. After 72 h in D (25C)activity increased in continuous light. Therefore, the regulatoryrole of light, nitrate and ammonium in the process of appearanceof GS was mainly studied between 72 and 120 h after transferfrom after-ripening to germination conditions (phase II of thepre-germination process). The inducing effect of red light ismediated by the photoreceptor phytochrome: the effect of long-termcontinuous red light (6 or 24 h) can be reversed, at least inpart, by a subsequent far-red light pulse (‘end of day’Irradiation). Blue light is more effective than red light ininducing the appearance of GS. Therefore, a specific blue lighteffect has to be assumed. This represents a novel mode of lightaction in regulating the level of the ammonium assimilatingenzyme in an angiosperm system. lmmunoblots showed that (i)increase in the enzymatic activity is caused by de novo synthesisof the enzyme protein, (ii) two different subunits (38 and 42kDa) contribute to the total activity which must be attributedto two different isofornis. In accordance with results fromother higher plants, the 38 kDa subunit (presumably relatedto the cytosolic isoform) did not increase in the presence oflight, whereas the 42 kDa subunit (presumably related to theplastidic isoform) was induced. The maximal enzyme level wasreached only in the presence of both light (blue light) andnitrate. Light induction was also observed in the presence ofammonium; however, GS activity was decreased, when comparedto nitrate-treated turions. Comparison of these results withprevious observations suggest that the influence of light andnitrate on the germination response and regulation of the nitrate/ammoniumassimilation pathway in turions appear to be unrelated phenomena. Key words: blue light, germination, glutamine synthetase, phytochrome, Spirodela polyrhiza, turion     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

Phytochrome-Mediated Increase in Glutamine Synthetase Activity in Photosensitive New York Lettuce Seeds

Red light increased glutamine synthetase (GS) activity in NewYork lettuce seeds before the initiation of axes elongation.The increase in GS activity was shown to be mediated by phytochrome.The escape reaction of the red-light effect on GS activity proceededfor several hours at 25°C, and roughly fitted that on thegermination. The effect of red light on the increase in GS activitywas inhibited completely by cycloheximide, but not at all byactinomycin D. The increased GS activity in the embryonic axesis discussed in relation to the promotive effect of red lighton lettuce-seed germination. (Received May 26, 1983; Accepted September 14, 1983)     

Blue and Red Light-dependent Alterations in the Ratio of two Forms of Glutamine Synthetase in Chlorella kessleri

Two forms of glutamine synthetase (EC 6.3.1.2) can be separated in crude extracts of Chlorella kessleri on the basis of their different surface charges. The two enzyme forms (GS1 and GS2) respond differently upon transferring the cells from darkness to autotrophic growth in white light: the activity of GS2 increases, that of GS1 remains unchanged. The increase in GS2 activity is only brought about by blue light; in red light GS2 activity appears to be uninfluenced, while that of GS1 increases. There are no indications of wavelength-dependent oligomerization processes as a cause for the observed activity alterations. There is however, a strong influence of inhibitors of protein biosynthesis. Cycloheximide and lincomycin both affect the blue light-dependent increase in activity of GS2, cycloheximide preventing that of GS1 in red lgiht completely. Since literature data point to localization of GS2 in the chloroplast, and GS1 in the cytosol, the data are discussed in view of two different photoreceptors involved in the regulation of the amounts of GS1 and GS2 in different compartments of the Chlorella cell.     

Factors involved in the coordinate appearance of nitrite reductase and glutamine synthetase in the mustard (Sinapis alba L.) seedling

The extent to which the appearances of nitrite reductase (NIR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) are coordinated was studied in mustard (Sinapis alba L.) seedlings. It was established by immunotitration that the increased activities of NIR and GS in the presence of light and nitrate can be attributed to the de-novo synthesis of enzyme protein. The bulk of the NIR and GS was found in the developing cotyledons. In the absence of nitrate in the growth medium there was no coordinate appearance of NIR and GS. While light strongly stimulated the appearance of GS, the level of NIR was hardly affected and remained low. On the other hand, in the presence of nitrate in the medium the appearances of NIR and GS were strictly coordinated, the GS level being considerably above that of NIR. It is argued that phytochrome-controlled synthesis of GS in the absence of nitrate is part of the mechanism to reassimilate ammonium liberated during proteolysis of storage protein and metabolism of the resulting amino acids, whereas the strictly coordinated synthesis in the presence of light and nitrate indicates the dominance of nitrate assimilation under these circumstances. The fact that the level of GS was always considerably above that of NIR appears to be a safety measure to prevent ammonium accumulation.Abbreviations FR standardized far-red light (3.5 W·m^–2), to drive the high-irradiance reaction of phytochrome - GS glutamine synthetase, EC 6.3.1.2 - NIR nitrite reductase, EC 1.7.7.1 This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation).     

Effect of Gibberellic Acid on Glutamine Synthetase Activity in Two Varieties of Lettuce Seeds, New York 515 and Grand Rapids

Gibberellic acid (GA) increased glutamine synthetase (GS) activityduring imbibition by lettuce seeds. The effect of GA was moreremarkable in the dark than under red light. The increase inGS activity induced by GA was inhibited completely by cycloheximide(1 mM). No promotive effect of GA on dark germination was observedin the presence of L-methionine-DL-sulfoximine, a specific inhibitorof GS. Therefore, GA has its promotive effect on the dark germinationof lettuce seeds mainly through GS activity. The GS activitypresent in the embryonic axes of Grand Rapids seeds was abouthalf that in New York 515 Improved, also the promotive effectof GA on dark germination was lower in Grand Rapids. This differencebetween dark germination in the New York and Grand Rapids seedswas interpreted as being the difference in the amount of GSactivity in the embryonic axes of the dry seeds. (Received September 29, 1983; Accepted November 29, 1983)     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

THE ROLE OF THE SEED-COATS IN GERMINATION OF PHOTOSENSITIVE LETTUCE SEEDS

1. In order to analyze the role of both the fruit-coat and theendosperm in the germination of Grand Rapids lettuce seed andin the elongation of the embryo, the seeds were subjected toa number of operations. The fruit-coat was found to make onlya minor con tribution to the photosensitivity of the seed. Thefact that 100% germination of these seeds can occur in completedarkness, when the restraining influence of the endosperm isremoved by appropriate cuts, makes clear that it is the integrityof the endosperm layer that lecides the photosensitivity ofthe seed to red and far-red light. Thus it is deduced that theeffect of light is exerted primarily on the mechanical propertiesof the endosperm layer.
2. Neither red nor far-red light wasfound to affect the elongationof the radicle of de-coated seeds,whereas both powerfully affectthe germination of intact seeds.It follows that radicle elongationis not itself the limitingfactor in germination, although ofcourse it follows immediatelyupon the germination process proper.
3. In order to account forthe ability of red light to initiategermination, it is proposedthat the final step in the germinationcontrol process is theproduction of an enzyme whose actionenables the tip of theradicle to penetrate through the coat.This makes it possiblefor the radicle to begin elongating.Although the detectionof such an enzyme in the radicle wasnot successful, the cotyledonsdo produce both a pectinase andan enzyme hydrolyzing carboxymethylcellulose.Furthermore, seedsinjected with a cellulolytic, hemicellulolyticor pectolyticenzyme germinated to nearly the maximum extentin the dark.This gives considerable support to the hypothesis.

¹ Present address: Johnson Foundation for Medical Physics, Universityof Pennsylvania, Philadelphia, Pa., U. S. A. (Received December 5, 1962; )     

Photosystem I and photophosphorylation-dependent leucine incorporation in the blue-green alga Anacystis nidulans

L-Leucine uptake and incorporation in the blue-green alga Anacystisnidulans were measured during illumination with monochromaticlight of 630 and 717 nm. With near as well as far red light,an enhanced uptake of ¹⁴C-L-leucine was observed. In far redlight, the leucine uptake depended on light intensity and pHvalue. After the first few minutes, the uptake remained constantfor more than one hour. The rate of uptake in light was thesame in air as in nitrogen. The incorporation of ¹⁴C-leucinein the soluble fraction decreased in the presence of chloramphenicolwhich prevents protein synthesis. In far red light, its incorporationwas insensitive to DCMU (5 ? 10^–6 M) but was depressedby uncouplers like CCCP or desaspidin. These effects are takenas evidence that leucine incorporation under the conditionsused is dependent on photosystem I reactions and cyclic photophosphorylation.DBMIB and KCN in high concentrations decrease the leucine incorporationin far red light and indicate that plastoquinone and plastocyaninare members of the cyclic electron flow also in intact cellsof Anacystis. Antimycin A has no inhibitory effect. The inhibitionby other less specific inhibitors like salicylaldoxime, desaspidinand DSPD is discussed. (Received August 19, 1978; )     

Light Actions in the Germination of Cocklebur Seeds: IV. DISAPPEARANCE OF RED LIGHT-REQUIREMENT FOR THE GERMINATION OF UPPER SEEDS SUBJECT TO ANOXIA, CHILLING, CYANIDE OR AZIDEPRETREATMENT

Esashi, Y., Fuwa, Nn Kojima, K. and Hase, S. 1986. Light actionsin the germination of cocklebur seeds. IV. Disappearance ofred light-requirement for the germination of upper seeds subjectto anoxia, chilling, cyanide or azide pretreatmenL—J.exp. Bot. 37: 1652–1662. The effects on the germination of positively photoblastic uppercocklebur (X anthium pennsylvanicum Wallr.) seeds by pretreatingwith anoxia, chilling, cyanide or azide, which stimulates theirdark germination, were examined in relation to light actions.Prior to experiments, seeds were pre-soaked at 23 °C inthe dark for 1 or 2 weeks to remove the pre-existing Pfr. Whenthe prctreatment conditions were suboptimal for germinationinduction, the stimulating effects of the pretreatments on germinationduring a subsequent dark period at 23 °C were manifest onlywhen seeds were irradiated with red light before or after thepretreatment Red light promotion was reversed by blue or far-redlight treatment. However, both prc-chilling for 6 d at 8 °Cand prctreatment with 1· 5 mol m ^{– 3} NaN³ for 2d could induce full germination without red light exposure.On the other hand, both pre-exposure to anoxia for 8 d and pretreatmentwith 30 mol m^–3 KCN could induce the dark germinationonly when germination occurred at 33 °C which is known toaugment the ratio of an alternative respiration flux to a cytochromeone. Moreover, the dark germination in response to these inductionswere strongly inhibited by the inhibitors of alternative respiration,propyl gallate and benzohydroxamic acid, applied during a subsequentdark period. It was thus suggested that Pfr has some relationto the operation of two respiration systems of cocklebur seeds,but it is not indispensable to germination of this positivelyphotoblastic seed. Key words: Anoxia, azide, blue light, chilling cyanide, dark germination, far-red light, red light, seed germination, X anthium pennsylvanicum     

Priming and accelerated ageing affect L-isoaspartyl methyltransferase activity in tomato (Lycopersicon esculentum Mill.) seed

Damage and degradation of cellular proteins is observed duringage-induced seed deterioration. L-Isoaspartyl protein methyltransferase(EC 2.1.1.77 [EC] ) is an enzyme hypothesized to play a role in limitingand repairing age-induced damage to proteins. Tomato (Lycopersiconesculentum Mill. ‘New Yorker’) seeds were assayedfor changes in L-isoaspartyl methyl-transferase activity duringaccelerated ageing and after osmotic priming. Accelerated ageingof seeds for 1–4 d at 45C and 100% relative humidityreduced germination from 94% to 71%, increased the mean timeof germination (MTG) from 2.4 to 5.8 d, and was accompaniedby a correlative decrease in L-isoaspartyl methyltransferaseactivity (r²=0.90). Aged and untreated seeds were primed for7 d at 20C in darkness using aerated solutions of 3% KNO₃ orpolyethylene glycol 8000 (PEG) with equivalent osmotic potential(–1.25 MPa). Priming with KNO₃ decreased the MTG, butdid not improve germination percentage for untreated seeds.Priming did not affect L-isoaspartyl methyltransferase activityin untreated seeds, but restored activity in aged seeds primedin KNO₃ to levels near that of untreated seeds. Priming withPEG did not effectively improve the MTG or increase L-isoaspartylmethyltransferase activity. During germination, L-isoaspartylmethyltransferase activity remained constant for 48 h post-imbibitionand then declined, suggesting that the enzyme was developmentallyregulated and inactivated or degraded as radicle emergence occurred. Key words: L-Isoaspartyl methyltransferase, protein repair, seed priming, accelerated ageing, Lycopersicon esculentum