INHIBITION OF GROWTH AND METABOLISM OF CHORELLA AND SOME OTHER PLANT TYPES BY CALCIUM DIPICRYLAMINE AND OTHER POISONS J.F. BIERHUIZEN NN08201.

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1 INHIBITION OF GROWTH AND METABOLISM OF CHORELLA AND SOME OTHER PLANT TYPES BY CALCIUM DIPICRYLAMINE AND OTHER POISONS J.F. BIERHUIZEN NN821.23

2 INHIBITION OF GROWTH AND METABOLISM OF CHLORELLA AND SOME OTHER PLANT TYPES BY CALCIUM DIPICRYLAMINE AND OTHER POISONS ERRATA p. 27 Legend t figure 13, fr: NH 4 OH.HCl, read: NH 2 OH.HCl p. 46 In table XIII, last clumn, fr: 7, read: 8 p th line frm belw, fr: 1" 2 M CaD 2, read: 1" 3 M CaD 2 p. 55 5e regel van nder, 1~ 2 M CaD 2, met zijn: 1~ 3 M CaD 2 Prefschrift J.F. BIERHUIZEN, Medcdeling Landbuw Hgeschl N. 57(7) Reprinted frm MEDEDELINGEN VAN DE LANDBOUWHOGESCHOOL TE WAGENINGEN/NEDERLAND 57 (7), 1-59 (1957) Labratry f Plant Physilgical Research, Agricultural University, Wageningen, Netherlands, 163th Cmmunicatin; 57th Cmmunicatin n Phtsynthesis (Received 27.5:57)

3 Dit prefschrift met stellingen van JOHAN FREDERIK BIERHUIZEN landbuwkundig ingenieur, gebren te Paree (Java), 8 ktber 1926, is gedgekeurd drde prmtr, Dr. E. C. WASSINK, hgleraar in het Plantenfysilgisch Onderzek en defysilgie der Planten. Wageningen, 27 mei De Rectr Magnificus der Landbuwhgeschl W. DE JONG

4 INHIBITION OF GROWTH AND METABOLISM OF CHLORELLA AND SOME OTHER PLANT TYPES BY CALCIUM DIPICRYLAMINE AND OTHER POISONS PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE LANDBOUWKUNDE OP GEZAG VAN DE RECTOR MAGNIFICUS IR. W. DE JONG, HOOGLERAAR IN DE VEETEELTWETENSCHAP, TE VERDEDIGEN TEGEN DE BEDENKINGEN VAN EEN COMMISSIE UIT DE SENAAT VAN DE LANDBOUWHOGESCHOOL TE WAGENINGEN OP VRIJDAG 5 JULI 1957 TE 16 UUR DOOR JOHAN FREDERIK BIERHUIZEN Bibli«4h4dek Ltndbuw Hlgi!>mchi WAGE N'NG^N H. VEENMAN & ZONEN - WAGENINGEN

5 STELLINGEN I Hewel calcium dipicrylamine k het maximale lichtrendement van de phtsynthese verlaagt, is aan te nemen dat deze remstf geen narcticum maar een specifiek enzym vergif is. Dit prefschrift. II Ofschn calcium dipicrylamine inzeerlage cncentraties remmend werkt p de plantengrei, is een pbrengstverlaging in het veld niet te verwachten bij gebruik van kali meststffen, waarin deze stf als verntreiniging aanwezig is. HI De cnclusie van VEIHMEYER en HENDRICKSON uit verdampingspreven met bnen, dat de verdamping bij een stijging van de vchtspanning in de grnd niet vermindert, is niet van tepassing nder nrmale cultuurmstandigheden. VEIHMEYER, F. J., A. H. HENDRICKSON, Trans. Am. Geph. Unin 36, p (1955). IV Een afdend bewijs vr het ptreden van kritische periden" ten aanzien van de waterbehefte bij cultuurgewassen is uit veldberegeningspreven znder meer niet af te leiden. V Het verdient aanbeveling de tepasbaarheid van de Anwelkmethde" als methdiek van nderzek k in Nederland vr de landbuw nader te bestuderen. ARLAND, A., Sitzungsber. Deutsch. Akad. Landwirtsch. Bed. 5 (6), p (1956). VI De huidige wijze van aardappelverkp biedt de huisvruw weinig waarbrg vr de kwaliteit. Het verdient aanbeveling in de aardappelhandel ver te gaan tt klein-verpakking enclassificatie van de waar naar gebruiksdel en kwaliteit. VII Gezien ns klimaat en de gebrekkige kennis mtrent de aard van de drgteresistentie, zullen de nederlandse kweekbedrijven de veredeling p drgteresistentie van gewassen, waarvan zaaizaad f ptged wrdt geexprteerd, vrshands ng in aride gebieden meten uitveren. VIII Bij de bepaling van de dagelijkse waterbehefte van landbuwgewassen vr irrigatiedeleinden met uit praktisch gpunt geen nderscheid gemaakt wrden tussen transpiratie en evapratie. ACHTNICH, W., Wass. u. Bd. 8, p. 398^1 (1956). Prefschrift J. F. Bierhuizen Wageningen 1957

6 WOORD VOORAF Hewel ditprefschrift ver remming" handelt, vel ikmij gedrngen, alien, die mijn wetenschappelijke vrming gestimuleerd" hebben en mij behulpzaam zijn geweest bij het bewerken van deze dissertatie, van harte dank te zeggen. U, hggeleerde WASSINK, hggeachte prmtr, dank ik in het bijznder vr het in mij gestelde vertruwen. Ik heb het steeds zeer gewaardeerd, dat ik p Uw labratrium in z ruime mate de gelegenheid vnd mij aan het nderzek te wijden. Uw hulp bij de bewerking van het manuscript zal ik niet licht vergeten. Zeer geachte Mevruw WASSINK-VAN LUMMEL, een wrd van dank vr Uw gede zrgen mag ik hier zeker wel aan tevegen. De N. V. MEKOG, IJmuiden, gaf mij de gelegenheid dit nderwerp te bestuderen, waardr ik mijn prefschrift kn samenstellen. Speciaal U, weledelgestrenge EBBINGE WUBBEN, ZOU ik ng willen bedanken vr de vele en vruchtbare cntacten, welkeik in verband met dit nderzek met U mcht hebben. Hggeleerde COOLHAAS, hggeleerde DORST, metgrte waardering denk ik terug aan Uw clleges tijdens mijn ingenieursstudie. Zeergeleerde SPRUIT, vr de waardevlle suggesties en Uw warme belangstelling ben ik U zeer erkentelijk. Beste WINTERMANS, vr het drnemen van het manuscript ben ik je zeer veel dank verschuldigd. Ten alle tijde was je bereid mij met raad en daad terzijde te staan. Het technisch persneel van het Labratrium vr Plantenfysilgisch Onderzek issteeds zeer behulpzaam geweest; hiervan wilik met name nemen de heer en mevruw KERKHOF-DE Vs, dr wie de cultures zeer accuraat werden verzrgd, terwijl de heer STEDELAAR nauwgezet zrg dreg vr de uitvering der tekeningen. Ik wil U alien hiervr hartelijk danken. Zeergeleerde VAN DEN BERG, znder Uw welwillende medewerking, wat betreft mijn tijdsindeling tussen remstffen en waterhuishuding, had ik mij deze laatste maanden niet z vlledig aan dit prefschrift kunnen wijden. De heer BERENDS ben ik erkentelijk vr de crrecties in de engelse taal. Meder, vl vreugde betuig ik U dr middel van dit prefschrift mijn dank vr de talls vele pfferingen, welke U zich, na vaders verlijden, vr de pleiding van Uw kinderen hebt getrst. Ans, je hebt me gedurende de gehele bewerking van het prefschrift met raad en daad bijgestaan; vral de laatste ldjes wgen zwaar. Ok ju, Elly, zu ik ng graag vr je vele hulp willen bedanken.

7 CHAPTER I. Intrductin CONTENTS 1. Scpe f the present investigatin 2 2. Sme remarks n literature n inhibitin, with special reference t phtsynthesis 3 P. CHAPTER II. Material and methds 1. Methd f cultivatin f the algae 5 2. Descriptin f the WARBURG apparatus 6 3. Measurement f the gas exchange 6 4. Measurement f the inhibitin 11 CHAPTER III. Inhibitin f phtsynthesis with CaD 2 as cmpared with ther inhibitrs in Chlrella 1. Intrductin Influence f inhibitr cncentratin, at light saturatin Influence f time after additin f the inhibitr, at light saturatin Effect f temperature, at light saturatin Influence f light intensity Discussin 27 CHAPTER IV. Inhibitin f phtsynthesis with CaD 2 as cmpared with the inhibitin f ther metablic prcesses in Chlrella 1. Intrductin Inhibitin f endgenus and glucse-stimulated respiratin Inhibitin f phtsynthesis at different ph f the medium The inhibitin f phtsynthesis in cnnectin with flurescence Inhibitin f phtsynthesis with CaD 2 in flashing light Inhibitin f auttrphic and hetertrphic grwth Discussin 4 CHAPTER V. Inhibitin f phtsynthesis and grwth with CaD 2 in varius plant types as cmpared with Chlrella 1. Intrductin Inhibitin in Lemna minr, Lemna gibba and Lemna arrhiza 42

8 2 57 (7) 3. Inhibitin in Scenedesmus Inhibitin in Chrmatiwn Experiments with Rhdspirillum rubrum, Oscillatria species, Nstc species, Chlamydmnas Discussin 49 CHAPTER VI. Discussin and Summary I. General discussin Summary Samenvatting 55 References 56 CHAPTER I INTRODUCTION 1. SCOPE OF THE PRESENT INVESTIGATION Hexanitrdiphenylamine r dipicrylamine (abbreviated as D) is a weak acid, which has a lw slubilih in water (.1 mg/1.). Salts f dipicrylamine are easily sluble in water with the exceptin f the ptassium salt. The slutins have an intensely red clur, while sdium and ammnium salts have been widely used as dyes fr leather and silk and fr light filters fr phtgraphic purpses under the name Aurantia". On accunt f the selective inslubility f the ptassium salt, dipicrylamine can be used as a reagent fr ptassium in spt tests (FEIGL [15]). Industrial applicatin f D has started with the separatin f ptassium ut f seawater (Kielland, Nrwegian patents). In cnnectin with research by the N.V. Mekg, the authr has made experiments n the influence f D n sme physilgical prcesses, especially phtsynthesis, in relatin t pssible cnsequences f pllutin f ptassium fertilizers by D. Only few indicatins f txic actin f D were available frm literature. It has been shwn that D in waste waters is txic fr fishes already at extremely lw cncentratins (59). GAVAUDAN et al (24) reprted a strng distrtin f cell divisin by D, even at lwer cncentratins than is fund fr clchicine. The wrk reprted here cnsiders especially the effects f D n phtsynthesis, respiratin and grwth in Chlrella, which was used as a simple test plant. Our experiments were perfrmed with the calcium salt f dipicrylamine, CaD2, since this salt is readily sluble and NO 2 NO, culd be maintained in slutins even NO a NO. fr a lng time withut an appreciable decrease in cncentratin. The experiments are mainly cncerned with inhibitin f varius metablic prcesses by D under different cnditins f temperature, light intensity, ph, etc. Mrever, experiments were made in which the effects f CaD-2 werr cmpared with thse f ther, well-knwn inhibitrs (Chapters III and IV).

9 57(7) 3 Other plant species have als been tested as t their reactins upn D in rder t find ut if their respnses are similar (Chapter V). Since D will always be applied tgether with ptassium, the ptassium metablism f the plant may be imprtant in this respect. Hwever, the lw cncentratin f D at which inhibitin f grwth and phtsynthesis already was bserved, and the different effects f ptassium starvatin and D inhibitin bserved in Lemna (Chapter V, see als [5]) decrease the pssibility f a direct cnnectin between D-effects and ptassium metablism. The latter, therefre, has nt been studied in greater detail. A brief review f sme literature n inhibitin f metablism in plants will be given in sectin 2f this chapter. 2. SOMEREMARKS ON LITERATURE ON INHIBITION, WITH SPECIAL REFERENCE TO PHOTOSYNTHESIS Inhibitrs affect many prcesses in plants; the nature f the respnse des nt nly depend n the cncentratin applied, but als n the different sensitivity f varius parts f the plants. It must be brn in mind that n applicatin f a pisn t intact plants r islated tissues, an verall effect is measured invlving the mvement and partitin f the substance between the pint f applicatin and varius sites f txic actin. The respnse t 2,4-dinitrphenxyacetic acid,fr instance, hasbeen shwn n phtsynthesis (FREELAND [19]), water relatins (BROWN [11]), stmatal behaviur (BRADBURRY and ENNIS [8]) and in absrptin and accumulatin (NANCE [4]), SMITH et cl. [63]). It was, therefre, felt as imprtant t use a simple test plant like Chlrellawhich is easier fr inhibitin treatments because f little differentiatin, while mass cultures are easily btained in simple nutrient slutins. In representing the percentage inhibitin against the lgarithm f the dsis, an apprximately linear relatinship is btained in several cases (AUDUS [3], BROWN and WEINTRAUB [12], WARBURG [71]) which is in accrdance with the adsrptin thery f FREUNDLICH (2). In many cases deviatins frm this relatinship ccur at threshld and near cmplete inhibitin s that a mre r less sigmid curve results. TAMIYA (68), studying inhibitin phenmena in phtsynthesis under different cnditins, derived a functinal relatinship between the degree f inhibitin (H) and the cncentratin f the inhibitr (G), accrding t the frmula: G n H = n i p n ( n anc * are cnstants representing the rder f inhibitry reactin and the cncentratin causing 5% inhibitin respectively). Hwever, deviatins frm this frmula still ccur, which may be ascribed t differences in behaviur f the individual cells, r may depend n the naturef the inhibitr. Cmparisns between cmpunds nly based n a single dse check may give misleading results,since, as stated abve,fr a large number fbilgical effects the relatinship between the effect and the dse is represented by a sigmid curve. The cncentratin giving 5 % inhibitin r mrtality established by applying a range f cncentratins, therefre, gives the mst reliable infrmatin in spite f the fact that threshld and cmplete inhibitin are smetimes mre imprtant data fr practical purpses. BLACKMAN (7), TAMIYA (68) and many thers,express the relatin in terms fequieffective dses.

10 4 57 (7) Phtsynthesis is generally mre affected by inhibitrs than respiratin, the latter usually being reduced nly at higher cncentratins. GAFFRON (21), hwever, reprted the reverse in Scenedesmus strain D, which prbably is the nly case hithert knwn. The same authr (23) cmpared the inhibitin f phtsynthesis with that f phtreductin in rder t find ut whether an inhibitr f phtsynthesis affects the xygen liberating system nly. WARBURG (71) already distinguished tw types f inhibitrs, v/z., specific enzyme pisns and narctics. Enzyme pisns act nly n certain specific enzyme surfaces, while narctics indiscriminately blck all enzyme surfaces. Several specific pisns have been fund which nly affect the light saturatin level f phtsynthesis while narctics decrease bth this level and the initial slpef the phtsynthesis versus light intensity curve. Hwever, the distinctin between these tw types is nt always clear. WELLER and FRANCK(8), e.g., bserved a decrease with hydrxyl amine under light limitatin, while GAFFRON (23) clearly shwed a specific enzyme inhibitin with this pisn in experiments n phtreductin in Scenedesmus. If the initial slpe in a light intensity versus phtsynthesis curveis changed upn additin f an inhibitr, a change in the initial slpe f flurescence, either increasing r decreasing, may be expected since interference with energy transfer may then be assumed t ccur. A decrease in flurescence yield will be bserved when the inhibitr itself has a high degree f energy acceptance. Cnsideratins alng these lines have been brught abut in extensive studies with Chlrella by FRANCK et al. (16,17,18) and with Chlrella, Chrmatium and diatms by WASSINK et al. (75,76, 77). The effect f D n flurescence, therefre, has been studied at different light intensities in rder t find ut whether the inhibitin with D bserved at light limitatin, als changes the initial slpe f the flurescence versus light intensity curve. In additin t this sme flashing light experiments were made, in rder t sustain the infrmatin btained in cntinuus light. TAMIYA (68), using varius inhibitrs, has bserved an increase as well as a decrease in inhibitin f phtsynthesis with a decrease in temperature. Cmparing the results f cyanide inhibitin n phtsynthesis and catalase activity, he nticed, hwever, that the temperature effect bserved in the last mentined inhibitin des nt always appear in phtsynthesis. He explained this by assuming that the reactin step which is suppressed by cyanide, becmes less rate determiningfr the verall phtsynthetic prcess at lwer temparatures. A temperature effect n applying sprays t insects has been reviewed by BROWN (9), wh ascribed this phenmenn t: 1. the rate at which, and the extent t which the pisn is absrbed by an rganism and reacts n itscells and tissues. 2. the rate at which, and the extent t which the pisn is eliminated by decmpsitin r detxicatin, excretin r washing ut. With rapidly acting pisns killing may be faster at higher temperatures, with less rapidly acting pisns the reverse may ccur; a recvery in general will be slwer at lwer temperatures. Experiments by POTTER and GILHAM (48) and by TAMMES (66) n animals pint t such relatinships. In cnnectin heret the attentin shuld be drawn t the surveys f LIPSCHITZ (37) and HEUBNER (28) n decmpsitin f armatic nitr- and amin cmpunds. Accrding t LIPSCHITZ (37) these cmpunds are metablized in animal tissues accrding t:

11 57(7) in which txicity may be indicated as R.NO2 H, H, R.NO^ + I± R.NH.OH < R.NH In this case sme f the intermediate prducts, especially the hydrxyl amine type, are txic. CHAPTER 11 MATERIAL AND METHODS 1. METHOD OF CULTIVATION OF THE ALGAE Chlrella, strain A, was used thrughut the assimilatin and respiratin experiments. The algae were kept as sterile stckcultures in tubes with 1.5% agar and a medium which per litre cntained: a. FeSO/ MgS 4 Na-citrate KNO3 KH 2 P 4.3 g g. l.oog g g. b. glucse 15 g. Slutins a. and b. were sterilized separately and mixed afterwards. The tubes were in a light cabinet after VAN NIEL, in which a cntinuus illuminatin was btained by a vertical rw f incandescent lamps. Ventilatin f the cabinet resulted in a temperature f abut 2 C, being the mst favurable temperature fr grwth f Chlrella. The cultures fr the experiments were prepared as fllws: The algae were inculated with a platinum wire int 1 cc flasks cntaining 5 cc liquid medium f the same cmpsitin as the agar medium. These cultures were als incubated in the light cabinet fr several days. In this way, by the stimulatingeffect f glucse, a rapid grwth was btained. Frm these cultures, 5 cc inculum were transferred t a ne litre erlenmeyer cntaining 5 cc f WARBURG culture medium. This medium per litre cntained: a. Ca(N 3 ) 2 KNO3 NaCl l.oog..25 g..15 g. b. KH 2 P 4.25 g. c. MgS 4.5 g. d. FeS 4.6 g. In additin 2 cc f an A 4 and B 7 slutin f trace elements accrding t ARNON (I) were supplied. Cncentrated stck slutins f the salts mentined abve, were sterilized separately (b, c, and d), r in a mixture f suitable matched quantities (a). The erlenmeyer flasks were placed n a shaking device and illuminated frm belw by 4 daylight flurescent tubes at light intensities frm lux n the bttm f the erlenmeyers placed n the machine. The agitatin served t prevent precipitatin f the cells and t ensure a hmgeneus illuminatin. The suspensin was flushed with a stream f air cntaining 5% C 2 thrugh a capillary tube clsed by a cttn plug. The average rm temperature was 23 C, the temperature f the suspensin, hwever, was 2-3 degrees higher dependent n duratin and intensity f illuminatin. Studies n inhibitin f phtsynthesis and grwth in varius ther plant types are presented in Chapter V. The preparatin f the cultures fr these experiments isdiscussed in that chapter.

12 57(7) 2. DESCRIPTION OF THE WARBURG APPARATUS Phtsynthesis and respiratin measurements were made in a WARBURG apparatus. The apparatus was f the usual type (figure 1), and had a thermstat bath f 1 x 3 cm, which culd be kept at temperatures between 15 and 35 C C with an accuracy f.5 C by an electric heater cntrlled by a therm relay and a cling system cnsisting f a cntinuus flw f tap water thrugh a cpper spiral. Tw Philra SO 1" sdium lamps under the glass bttm f the thermstat were munted in a white reflectr; the lamps were cled by tw small (25W) fans. A shaking frame n which 12 manmeters culd be placed, was munted in frnt f the thermstat. This frame is mved via an excentric at a rate f 15 revlutins per min. with an amplitude f abut 5 mm. The stirring f the waterbath was achieved by a hrizntal stirrer with 6 blades. The cnical vessels with flat bttms had a vlume f abut 2 cc, including tw side arms f abut 3-4 cc. FIG. 1. Frnt view f the WARBURG apparatus. Different light intensities were btained by filters, placed in metal hlders fixed nt the vessels. The light intensities were measured by a phtcell which was calibrated against a large surface thermpile (KIPP en Zn., Delft). The psitin f the phtcell was such that its sensitive surface culd be placed at the same level as the bttm f each vessel. The highest light intensity btainable in the arrangement was abut 6, ergs/cm 2 sec. 3. MEASUREMENT OF THE GAS EXCHANGE Measurements with the WARBURG apparatus are based n the manmetric principle, which implies that nly thse reactins can be bserved, in which gases are frmed r disappear. Frm the verall equatin f phtsynthesis: C 2 -f H..O -> CH it fllws, hwever, that fr each C 2 mlecule cnsumed, ne mlecule f 2 is released which implies that n net gas exchange at all wuld be apparent. This difficulty can be vercme by varius methds, which all have their advantages and disadvantages. This will be discussed in sme detail belw.

13 57(7) /. Measurements in which CO* is kept at a cnstant tensin by a buffer mixture. It is readily understd that the change in manmetric reading (h) is due slely t xygen evlutin r uptake if the partial pressure f carbn dixide in the gas space f the reactin vessel is maintained cnstant. Mixtures f carbnates and bicarbnates have been intrduced by WARBURG (72) fr keeping cnstant the partial pressure f CO2. A large uptake f carbn dixide frm these mixtures, hwever, decreases the carbn dixide pressure, especially if nly small amunts f bicarbnate are present. Lwer suspensin densities wereused r shrt time experiments were perfrmed if such a decrease culd be predicted. Usually mixture n. 9, cnsisting f.15 ml. carbnate and.85 ml. bicarbnate (ph 9.8),wasused in experiments n phtsynthesis. Buffer n. 9 is in equilibrium with a carbn dixide cncentratin f 9.1 X 1-5 M at 25 C. This cncentratin is nt a limiting factr in phtsynthesis f Chlrellaas was already bserved by WARBURG (72), wh reprted a strng decrease in rate f phtsynthesis nly at CO2 cncentratins lwer than ne tenth f that maintained by mixture n. 9. Hwever, the ph and the catin cncentratin f the buffer may have an effect n phtsynthesis as well. Figure 2 represents the rate f phtsynthesis at different light intensities measured in mixtures n. 9 and n. 2 (ph 1.3, 1. X 1 6 M C 2 at 25 C). It shws that the lwer rate f phtsynthesis in buffer n. 2, with the lwer carbn dixide pressure, als ccurs under light limitatin, which wuld nt be expected theretically. This pints t sme disturbing effect f high ph, apart frm a pssible limitatin f phtsynthesis by the lw carbn dixide cncentratin. In rder t retain the advantage f a cnstant CO2 pressure prvided by the WARBURG buffer, whilst reducing the trublesme ph-effect, Chlrella was suspended in distilled water while.2 ml. buffer was placed in the side arms. The slw diffusin rate f CO2 frm the buffer t the suspensin in the main part f the vessel, hwever, made it impssible t use nrmal suspensin m m" 2 /hu x 1 ergs/cm 2 FIG. 2. Rate f phtsynthesis f Chlrella in WARBURG buffer n. 2 (-i r) and n. 9 ( ), at different light intensities, at 25 C C (ne ut f 3 experiments). sec.

14 8 57(7) densities. N limitatin bythe lw ratef carbn dixidediffusin was bserved when nt mre than 1-2 mm 3 f cells were used per flask. Nw, under cnditinsflight limitatin, the same rate f phtsynthesis is fund in buffers n. 9 and n. 2, as is shwn in figure 3. Thugh this statement depends n ne pint nly, the cnsistency f the results f 3different experiments justify this deductin. Thus, thelwer ratef phtsynthesis bserved at light limitatin may be attributed t an unfavurable effect f the high ph fbuffer n. 2, in case Chlrella is suspended directly in the buffer. mm 2 / hur 3 r x1 4 ergs/ cm 2 sec. FIG. 3. Rate f phtsynthesis f Chlrellain tap water at different light intensities with WARBURG buffer n. 2 (+ +) and n. 9 ( ) in the side arms (ne ut f 3 experiments). PRATT (51) bserved that sdium carbnate bicarbnate mixtures had a depressive effect n phtsynthesis in lng term experiments, while ptassium mixtures had an accelerating effect. In a slutin in which the mlar rati f sdium t ptassium was 65/35, the increasing and decreasing actins f the ptassium and sdium salts respectively were balanced, and accrding t PRATT the initial rate f phtsynthesis was virtually cnstant fr fifteen hurs. In ur experiments, we have nly used sdium carbnate and bicarbnate mixtures in rder t avid a pssible precipitatin f D by ptassium. The gas exchange (x) was calculated in the well-knwn way by multiplying the measured pressure change (h) by the vessel cnstant (Vc) x = h x Vc 273 Vg x + Vfc while Vc = 1, Herein Vg represents the vlume f the gas space in the vessel, the capillary tube and the manmeter, Vf the vlume f the liquid phase and a the absrptin cefficient fr C 2 at temperature T. Usually, 2ccf algal suspensin wereusedin experimentsnrespiratin and phtsynthesis. As the ttal cntent f the vessels was abut 2 ml., the vessel cnstant fr xygen was near 1.65 at 25 C C. One r tw f the manmeter vessels were prvided with suspensin liquid nly, and served as therm-barmeters.

15 57 (7) 9 //. Simultaneus measurements f CO2 and >z were made by using the tw vessel methd. Identical samples f algae suspended either in 2 r in 5 cc f the same suspensin liquid under identical cnditins f light intensity and temperature, may be assumed t have equal gas exchange. This, hwever, will cause unequal pressure changes in bth manmeters, wing t the different slubilities f C 2 and O a in the suspensin liquid. Frm the pressure changes f the tw manmeters, the gasexchange (mm 3 ) can be cmputed by the frmula hkc, - HKc, hkp, - HKQ, ' t kcq, Kc, k c, kp, _ Kp a k a K, kc 2 Kc 2 in which h is the manmetric reading f the 2ccvessel in mm, H is the manmetric reading f the 5ccvessel in mm, kc, and Kc t are thevessel cnstantsfr C 2 (p.8)fr the 2cc and 5ccvessel respectively, k 2 and K a are the vessel cnstants fr 2 fr the 2cc and 5 ccvessel respectively. Jn rder t prvide sufficient amunts f C 2 fr phtsynthesis, the suspensin and the gas phase were saturated with a mixture f 95% air and 5% C 2 by flushing the WARBURG vessels with the mixture in the dark fr half an hur. During this perid, the vessels were shaken in rder t attain equilibrium between gas phase and liquid. The suspensin had been aerated previusly with air and 5 % C 2 during a quarter f an hur. ///. Measurements with the ne vessel methd if the phtsynthetic qutient P = -O2ICO2 is knwn. This is in fact an experimental simplificatin f the freging methd. The gas exchange is determined accrding t the frmula hk* x kc. c a - h x Vc -- pkc, - k* In figure 4, Vc has been cmputed fr 2, 4 and 6cc fluid/flask at different assumed phtsynthetic qutients fr a flaskwith a ttal vlume f 2cc at 25 C C. Frm this it is clear, that an errneus value f p has a smaller influence with 6cc liquid than with 4 r2cc. In ur experiments, when using this methd, 5ccwas taken as the mst suitable quantity, since the danger f disturbing effects due t mixing f the fluid in the main flask and that in the side arm then still wasvery small. Vc FIG. 4. The influence f the phtsynthetic qutient p at 25 C n the change in Vc fr 2cc ( ), 4cc ( ) and 6cc (+ +) fluid per vessel (ttal vlume f thevessel 2 cc). f P

16 1 57(7) Measurements with the ne and the tw vessel methd have been made in tap water and in phsphate buffers. Phsphate buffers may shw carbn dixide fixatin r retentin accrding t: Na 2 HP 4 -f C 2 + H, t NaH 2 P 4 + NaHC 3, which retentin appears abve ph 5, and increases expnentially with increasing ph. Thismay resultin large errrsf the cmputed gas exchange. These errrs can be eliminated by direct determinatin f the retentin. Fr this purpse an amunt f a strng acid isadded frm the side bulb at the start f the experiment and in anther vessel at the end f the experiment, which acid will release the carbn dixide frm the buffer. JOHNSON (31) has suggested a methd f crrecting C 2 retentin, withut the additin f acid. Since at high ph values HCOJ as well as C 2 is retained in the slutin, the effective value f a (which will be designated as a x )willbe larger than the theretical value.jf thevessel cnstant is calculated by the usef ac u instead f a, retentin fco>is crrected autmatically. As a t, increases rapidly with increasing ph, the retentin crrectin becmes very large at ph values abve 7, and the accuracy f the C 2 measurements suffers accrdingly. The extent f the crrectin is cmputed accrding t the frmula: 2i = antilg (ph ) + 1 a Figure 5 represents the decrease in Vc (crrected with ^)with increase in ph f the buffer, cmputed fr nevessel. It is clear, that large errrs ccurif n crrectin fr a is applied. Vc 1 - ph 8 FIG. 5. The cmputed decrease in Vc versus increasing ph f phsphate buffers fr 2cc fluid/vessel (p - 1.6, ttal vlume = 2 cc, at 25 C C). T check the validity f these cnsideratins fr ur experimental cnditins, experiments were made at ph 7.2, 6.5,and 5.9. The gasexchange was cmputed withut crrectin, by adding acid, and als by using the abve equatin. The results are summarized in table 1, averaging three experiments. This table shws that withut applying the suggested crrectin large errrs in the cmputatin f the gas exchange ccur, especially at high ph values. Cmparing the acid additin methd with the methd f theretical crrectin, the last methd yielded lwer values at ph 7.2 and higher nes at ph 6.5 and 5.9. These differences may be due t uncertainties in the ph measurements which were made at the end f the experiments nly. Fr a fully critical check measurements t within.1 ph unit shuld be made during the experiments.

17 57(7) II TABLE I. The rate f phtsynthesis in Chlrella (mm 3 2 /hr.) in phsphate buffers at different ph values, as an average f 3 experiments, at 25 C C, under light saturatin (5-6 x 1 4 ergs/cm 2 sec). PH Withut crrectin Applying JOHNSON'S frmula Measured by additin f acid MEASUREMENT OFTHEINHIBITION Standard slutins f the inhibitrs were made,.5 t.2 ml.f which were pipetted int the sidearm f a manmetervessel, ultimately ascertaining varius inhibitin percentages. Usually, ne r tw cntrl vessels received.2 ml. f the slvent nly. The prcedure feach experiment included measurement f the nn-inhibited phtsynthesis in all flasks during half an hur. Only series frm which the cmputed gas exchange in all vessels shwed less than 5 % variatin were used fr inhibitin experiments. After half an hur different cncentratins f the inhibitr were tipped int themain spacef the vessels. After 1 minutes, when temperature equilibrium was re-established, the measurements were cntinued fr ne r mre hurs. Dependent n the rate f pressure change, manmeter readings were taken at intervals f 1 r mre minutes. The inhibitin is expressed by indicating the decrease in rate as a percentage f the cntrl. Usually, fr the sake f simplicity, the standard errrs have nly been indicated at the calculated inhibitin percentages, while the abslute values have been reprted withut reference t the standard deviatin. A cmment has t be made n the ageing effect, which ccurs in cultures f unicellular algae (VAN HILLE [29], WASSINK and KATZ [75], PRATT [5]) and which accrding t PRATT (49, 5) is due t a prductin f a grwth inhibiting factr, which shuld act like a catalist pisn rather than like a narctic (PRATT [49], WASSINK and KATZ [75]). Figure 6a represents the decline f phtsynthesis in Chlrella with age, under light saturatin, each pint representing an average f 5 experiments. The results are in general agreement with thse f WASSINK and KATZ and f PRATT. It isclear frm figure 6b that the inhibitin percentage depends n the agef the culture,especially at higher cncentratins f the inhibitr. Therefre, thrughut the experiments described furthern in this paper, nly cultures f 3 and 4 days ld were used in phtsynthesis experiments, unless indicated therwise. In table II, the rate f phtsynthesis and the inhibitin percentage at cncentratins f 1., 2.83, 5.2, and 1. X 1 6 M CaD2 are presented as a functin f different densities f suspensin. The rate f phtsynthesis is linear up t 2 mm 3 cells/flask and there is n significant influence f densityf the suspensin upn the degree f inhibitin.

18 12 57(7) ' mm /hur K^+ + A mean errr I % inhibitin 9 - B 7 mean errr days after inculatin FIG. 6a. The effect f age n the rate f phtsynthesis f Chlrella at 25 C C, under light saturatin ( x 1 4 ergs/cm 2 sec.) in WARBURG buffer n. 9. FIG. 6b. Theeffect fage n theinhibitin f phtsynthesisby CaD 2 ( = 1. x 1~ 5 M, x 1-6 M, x x x 1~ 6 M, and Q - 1. x 1" 6 M CaD 2 ). TABLE IF. The inhibitin f phtsynthesis in Chlrella, at different cncentratins f CaD 2, with different suspensin densities, as an average f 4 experiments, at 25 C, under light saturatin (5-6 x 1 4 ergs/cm 2 sec.),in WARBURG buffer n. 9. mm 3 Cells/ vessel Rate f phtsynthesis f the cntrl (mm 3 2 /hr.) 1. Per cent inhibitin at different cncentratins f CaD 2 (1~ 6 M) j ± 5 7 ± 2 11 ± 2 13 ± 2 8 ±2 5 ± 5 38 ± 4 41 ± 1 36 ± 1 37 ± 4 7 ± 3 58 ± 3 57 ±4 58 ± 4 52 ±4 78 ± 3 71 ± 3 73 ±4 74 ± 2 72 ±4

19 57(7) "> J CHAPTER III INHIBITION OF PHOTOSYNTHESIS WITH CaD 2 AS COMPARED WITH OTHER INHIBITORS IN CHLORELLA 1. INTRODUCTION As has been set frward in Chapter I, varius authrs have studied inhibitin f phtsynthesis in Chlrella and ther small algae. Different pinins have been expressed regarding the mdef actin f pisns, and the results btained with any special pisn d nt always agree. Especially, the distinctin between the type f actin f narctics" and,,specific pisns" is nt always clear. Mrever, differences in sensitivity twards the same inhibitr have been frequently bserved, als in experiments with the samespecies. The cultivatin f the algae may play an imprtant part in the bserved differences in inhibitin. The age f the culture als influences inhibitin (29, 5, 75; fig. 6b). It may be assumed that the methd f cultivatin either increases r decreases the amunt f certain catalists, r causes a change in the structure f the phtsynthetic apparatus r in the permeability f the prtplast. All such changes will affect the inhibitin f phtsynthesis upn administratin f a certain pisn. The pssible influence f cultivatin induced us t cmpare the inhibitin f phtsynthesis in Chlrella with CaD-2 with that f ther inhibitrs. This als may seem the mst reliable methd t find ut whether the actin is specific, wing taffinities t certainenzymesf the phtsynthetic apparatus, r whether it is a result f surface activity nly. 2. INFLUENCE OF INHIBITOR CONCENTRATION, AT LIGHT SATURATION 2.1. Calcium dipicrylamine The inhibitin f phtsynthesis by calcium dipicrylamine (CaD-z), is shwn in figure7a. The inhibitin is represented as a percentage f the crrespnding % inhibitin 1 r- #12.5 C 17.5 C #25 C k 2 r7 rs 1 1 Fig. 7a. Fr explanatin, see p.14. J L J 1 ' I i I J L 1 i t i i 1 M CaD 2 1

20 14 57(7) / Q inhibitin 1 r J L J I I f I 1 I r r 1 t f t f I Fig.7b M Ethyl urethane / Q inhibitin 1 r J L J I L jl J L J L_JL J-JL ' ' r4 1 r3 1 Fig.7c. M Phenyl urethane F G. 7. The inhibitin f phtsynthesis f Chlrella under light saturatin ( x 1 4 ergs/cm 2 sec.)in WARBURG buffer n. 9 with: a. CaD 2 at 25, 17.5 and 12.5 C C, d. hydrxyl amine at 25 C C, b. ethyl urethane at 25 C, e. 2,4-dinitrphenl at 25 C, c. phenyl urethane at 25 C C, f. sdium azide at 25 C C.

21 57(7) 15 / Q inhibitin 1 r Fig. 7d. J L I f.. f I J t_j r4 1 M Hydrxyl amine / Q inhibitin 1 i Fig.7e. i- 4 J L * I i t t i I J 1 1 f -3 1 M 2-4 Dinitrphenl % inhibitin 1 r Fig. 7f. J L ' I I I I t i X^i^MA«M^M«iL«MlH^iMi r4 1 r3 1-2 M Sdium azide

22 57(7) 16 cntrl against the mlar cncentratin f CaD2 n a lgarithmic scale, as an average f 5 experiments. The measurements have been made in the carbnate bicarbnate buffer n. 9 at light saturatin (48,-55, ergs/cm 2 sec), and at 25 C. Further details f experimental cnditins have already been described in Chapter II. The inhibitin increases regularly with an increase in cncentratin. The results are in accrdance with the adsrptin thery f FREUNDLICH (2) since nearly straight curves were btained if the cncentratin is pltted n a lgarithmic scale. The threshld f inhibitin is at abut 1 6 M. Cmplete inhibitin shifts t lwer cncentratins with increasing duratin f expsure t the inhibitr. A statinary state f inhibitin was hardly bserved after 2 hurs especially at the higher cncentratins. This fact will be discussed in greater detail in sectin 3 f this chapter. The results represented in figure7a are thse in which apprximately a statinary state was achieved. Cmplete inhibitin ccurred at abut 2 X 1 5 M CaD, as may be seen frm the figure. In higher CaD2 cncentratins, negative readings were bserved, exceeding thse f the cntrl in the dark. Cnsequently, the release f O2 was reversed in an uptake in the light. It seems likely that this is due t a phtxidatin. Anyhw, accrding t WARBURG this phenmenn may be taken as ein sicheres Zeichen der definitiven Zerstrung" Ethyl methane The urethanes have been intrduced as inhibitrs by WARBURG (71), wh reprted an increase in inhibitin with the increase in length f the C-chain accrding t TRAUBE'S rule. TRAUBE (69) bserved a cmplete crrelatin between the last mentined increase and the increase in capillary activity. Experiments by WARBURG (71) and bywassink et al. (78) pinted t the same inhibitin under light limitatin as well as under light saturatin. In figure 7b, the inhibitin f phtsynthesis with ethyl urethane (C2H5NHCOOC2H5) is given in per cent f the crrespnding cntrl. The inhibitin ccurs in the same range as was reprted by WARBURG (71) and by WASSINK et al. (78) with a threshld at 2. X 1* 2 M and cmplete inhibitin at 5. X 1 1 M. The inhibitin becmes statinary within 2 minutes and remains s fr at least 2 hurs after additin f the inhibitr. There is a direct relatinship between lg cncentratin and percentage f inhibitin, the curve, hwever, shws a peculiar bend between.1 and.2 M. It is pssible that in this range shrinking f the prtplasm wing t a desiccating effect by the smtic value f the slutin at these high cncentratins als plays a rle in the inhibitin. This is supprted by GREENFIELD'S bservatin (27) that the rate f phtsynthesis in strng light is inhibited by sucrse at cncentratins f.1 t.3 M Phenyl urethane Phenyl urethane (C6H5NHCOOC2H5) is nt sluble in water in cncentratins at which inhibitin ccurs. The use f.2% ethanl as a slvent was sufficient t disslve phenyl urethane insuch cncentratins as t btain 1 % inhibitin f phtsynthesis. N effect f.2% ethanl n phtsynthesis was bserved as may be seen frm table III.

23 57(7) 17 TABLE III. The inhibitin f phtsynthesis in Chlrella with ethanl as an average f 3 experiments, at 25 C C, under light saturatin (5-6 x 1 4 ergs/cm 2 sec.) in WAR BURG buffer n. 9. Cncentratin f ethanl in per cent Rate f phtsynthesis in mm 3 2 /hr Per cent inhibitin The inhibitin with phenyl urethane is represented by figure 7c as an average f 4 experiments. The inhibitin becmes cnstant within 2 minutes while threshld and cmplete inhibitin are at 4 X 1 5 and 3 X 1 3 M respectively. These results are in agreement with thse f WARBURG (71) and WASSINK et ah (78) Hydrxyl amine Hydrxyl amine (NH2OH.HCI) is an inhibitr strngly affecting phtsynthesis as was shwn fr the first time by SHIBATA and YAKUSHII (6). Similar results were btained by NAKAMURA (39), GAFFRON (23), WELLER and FRANCK (8), and TAMIYA (68). In his experiments with hydrgen adapted algae, GAFFRON (23) bserved a much smaller sensitivity f phtreductin. He cncluded frm this bservatin that the mde f actin f hydrxyl amine was as an enzyme pisn rather than as a narctic. Since phenyl urethane and ethyl urethane are knwn t behave like narctics (WARBURG [71], WASSINK et al [78]), hydrxyl amine therefre was als tested. The inhibitin is represented by figure 7d as an average f 4 experiments. It is clear that the curve is steeper than that btained with ethyl urethane and phenyl urethane. In this case the straight relatinship between lg cncentratin and percentage f inhibitin is nly evident between 2 and8 per cent inhibitin. Beynd these values the curve appraches assympttically and 1 per cent inhibitin. A cnstant inhibitin was bserved after 3 t6 minutes. The range at which inhibitin f phtsynthesis (in ur experiments 5% at 1.1 X 1 4 M) was manifest, was similar t that reprted by TAMIYA (68) ,4'Dinitrphenl Dinitrphenl ((NC^CeHaOH) is f interest in the present investigatin, because mre is knwn f this inhibitr in relatin t phtsynthesis (GAFFRON [23], WINTERMANS [83]), and because its structure mre r less crrespnds t that f CaD 2. As this inhibitr is mre sluble in alkaline slutins, standard slutins were made using WARBURG'S carbnate bicarbnate buffer n. 9 (ph 9.2) as a slvent instead f water. Accrding t WINTERMANS (83) the inhibitin is less prnunced in alkaline slutins, due t the fact that its activity is ascribed t the undissciated mlecules, because nly thse are able t penetrate int the cells. The inhibitin with 2,4-dinitrphenl in WARBURG buffer n. 9 (ph9.2) is represented in figure 7e, as an average f 4 experiments. The curve is steeper

24 18 57(7) than that fr phenyl urethane and ethyl urethane, and resembles mre that fr CaD 2 and hydrxyl amine. It is, therefre, mre difficult t indicate the cncentratins which represent threshld and cmplete inhibitin. As may be seen frm the figure, the cncentratin at which 5 per cent inhibitin ccurs is 3.8 X 1 4 M Sdium azide The inhibitin f phtsynthesis with sdium azide (NaN 3 ) is represented by figure7f, as an average f 4 experiments. The curve is less steep than that fr 2,4-dinitrphenl, CaD 2 and hydrxyl amine. Threshld and 1 per cent inhibitin are bserved at 1 X 1-4 M and 5 X 1 2 M respectively. 3. INFLUENCEOFTIMEAFTER ADDITION OFTHEINHIBITOR, AT LIGHTSATURATION Frm the preceding experiments it is clear that, thugh there was a slight decrease with time in the rate f phtsynthesis als in the blank, after additin f CaD 2 this rate drpped much mre clearly with time, especially at higher cncentratins. Figure 8gives anther example f this phenmenn. The lwer cncentratins shwed a tendency t remain cnstant arund 1.55 X 1 6 M CaD 2 ; belw that cncentratin a slight decrease in inhibitin with time was manifest. / Q inhibitin 1 x 5.x1 M 1 X1 5 M 8 JW i -K 5.2x1" 6 M 6 4 r8 x 3. X1 M 2-6 tj-1.55x1 M f rs +_^ ll.oxlo M hurs after additin FIG. 8. The inhibitin f phtsynthesis f Chlrella versus time after additin f different CaD 2 cncentratins at 25 C, under light saturatin ( x 1 4 ergs/cm 2 sec.)in WARBURG buffer n. 9, and the effect f washing n the inhibitin (f additin f CaD 2, \ time f washing).

25 57(7) 19 E(rel. units) & FIG. 9. The absrptin spectrum f calcium dipicrylamine in relative units. At least tw different explanatins f the increase in the inhibitin versus time after additin f CaD2 might be suggested, ne f which culd be alng theline f a phtdynamic actin. An absrptin spectrum f CaD2, therefre, was measured with a duble mnchrmatr (fig. 9). The data fr the emissin f the sdium lamp were taken frm ORANJE (43) and are represented in table IV. TABLE IV. The emissin f a sdium tube in different spectral areas in relative units (data frm ORANJE [43]). The line drawn between 5149/54 and 5683/88 represents the cut ff by the OG2filter.The wavelengths abve this line are cmpletely absrbed by the filter. Wavelength (A) Relative units 4391/ / / / / / / /88 589/ / n 1 Except fr the yellw lines at A, the emissin frm the sdium lamp is fairly strng in the infra-red and very weak in the blue-green spectral

26 2 57 (7) regin (table IV). CaD2 shws n absrptin at all in the yellw and infra-red, but a high absrptin in the blue-green spectral area (fig. 9). There is a slight pssibility, therefre, that the weak blue-green emissin frm the sdium lamp had a phtdynamiceffect n thecells, wing t absrptin bycad-2. Sme experiments have been made in which the inhibitin upn 9.9 X 1 6 M CaD2 was measured at the same time in different flasks. In sme f these flasks the blue-green radiatin was filtered ut by means f an OG2 filter which absrbs the blue-green rays cmpletely, as represented in table IV. The average inhibitin during the first hur after additin f CaD in five different experiments with 9.9 X l(hm CaD 2 was: with OG2 56 % ± 2 withut filter 58 % ± 2 The inhibitin was nearly the same in bth cases. Mrever, the inhibitin increased in the same way with and withut the OG2 filter. We may cnclude, therefre, that n phtdynamic actin can be respnsible fr the bserved time effect. The secnd suppsitin is that after additin fcad>n immediate equilibrium is established between the utside cncentratin and that in the cell, but that there is a cntinuus uptake f CaD. Hwever, the independence f the inhibitin f the glucse-stimulated respiratin frm time (Chapter IV, p. 32, figure 16) indicates that this suppsitin is prbably nt true. Washing experiments were made with three different cncentratins in rder t btain mre data n this phenmenn. These experiments cnsisted in measuring the inhibitin in several flasks with the same cncentratin fr ne hur. Halfthe numberf flasks were thencentrifuged and thealgaewashed twice with the suspensin liquid (WARBURG buffer n. 9) and then measured again in the WARBURG apparatus. It appears (fig. 8) that after washing n further increase f inhibitin ccurred while such increase was bserved in the flasks in which the CaD2 slutin had nt been remved by washing. The inhibitin in the washed algae, hwever, did nt decrease. Cmparable results were btained in ther experiments in which different cncentratins (3. X 1-6, 5.2 X lo 6 and 1. X 1 5 M) f CaD 2 were used. Tw imprtant cnclusins may be drawn frm these washing experiments: a) The increase in inhibitin with time is due t a cntinuus uptake f CaD2. b) The inhibitin is nt reversible. It is remarkable that the first cnclusin is nt supprted by the bservatin that the inhibitin f the glucse-stimulated respiratin remains unchanged with time (Chapter IV). In the latter case a cnstant degree f inhibitin was reached within 2 minutes after additin f CaD 2 (fig. 16), which time was mre r less the same as that bserved in inhibitin f phtsynthesis with the urethanes and hydrxyl amine. It is pssible that CaD2 is cntinuusly reduced, nly in light, t a prduct inhibiting phtsynthesis, while CaD 2 uptake can prceed nly as lng as this reductin takes place. This cnsideratin was tested, taking a cncentratin fcad> giving cmplete inhibitin after a lng time (fig. 8,5.2 X 1 6 M CaD2). This cncentratin was tipped at the same time int the suspensin in different WARBURG vessels. Hereafter, the inhibitin f phtsynthesis under light saturatin was measured, whereas smevessels remained in the dark and were illuminated ne and tw hurs later (figure 1).

27 57(7) 21 / Q inhibitin 1 r hurs after additin f CaD 2 FIG. 1. Inhibitin f phtsynthesis f Chlrellaversus time after additin f 5.2 x 1~ 6 M CaD 2 at 25 C C, under light saturatin ( x 1 4 ergs/cm 2 sec.) in WARBURG buffer n. 9 ( = 3 hurs in the light, + + = ne hur in the dark fllwed by 2 hurs in the light, = tw hurs in the dark fllwed by ne hur in the light). The inhibitin increases with time, aswas already mentined. It is clear that, after three hurs, the inhibitin f phtsynthesis was larger, when the time f illuminatin was lnger. Apprximately, cncurrent curves were btained in this way. This fact strngly supprts a cntinuus uptake f CaD2in light nly. These resultswillbediscussed with thse n the inhibitin f glucse-stimulated respiratin in Chapter VI. 4. EFFECT OF TEMPERATURE, AT LIGHT SATURATION In sme incidental experiments we fund that the inhibitin f phtsynthesis with CaD2 increases at lwer temperatures. Cnsequently, lwer cncentratins had t be used t btain the same range f inhibitins. TAMIYA (68) reprted an increase in the inhibitin f phtsynthesis with sdium azide and a decreasewith hydrxyl amine at lwer temperatures. Therefre, ur results with CaD2 btained at 25 C were extended t ther temperatures. The effect f temperature was investigated with ther inhibitrs als. In rder t btain results f the best pssible cmparability, the bservatins at different temperatures were made, if pssible, with the same culture in ne

28 22 57(7) day, s that differences in age f the algae culd be neglected. The temperature cefficient, Q\Q in ur experiments was 1.8 in the range frm 12.5 C t 25 C. The inhibitin f phtsynthesis with CaD2, ethyl urethane, phenyl urethane and hydrxyl amine is represented in table V as an average f 4-6 experiments. The results with CaD2 (table V) have als been pltted in figure 7a. Thugh there is a cnsiderable increase in inhibitin with CaD2 at lwer temperatures, the slpe f the inhibitin curve against lg cncentratin remains the same (figure 7a). The bserved increase in inhibitin is prbably nt effected by a higher uptake f CaD2 at lwer temperatures, as these figures were btained at equilibrium. Besides, the time t attain equilibrium was nearly the same at all temperatures. The influence f temperature n the inhibitin is given in figure 11. The cncentratin prducing 5% inhibitin is represented n a lgarithmic scale. In this way apprximately a straight line relatin is btained between temperature and cncentratin. C 3 r- 2 1 J I i i t i I., I i t t i 1 ri *- *-5 1 ' 1 w 1 M CaD 2 FIG. 11. The CaD 2 cncentratincausing5 %inhibitinf phtsynthesisversustemperature. With hydrxyl amine, phenyl urethane and ethyl urethane n the ther hand, neither a significant increase nr decrease f the inhibitin with temperature were bserved. 5. INFLUENCE OF LIGHT INTENSITY In previus years a cmprehensive study (52,53) has been made n the influence f light intensity and temperature n phtsynthesis. Generally, a linear relatin between light intensity and phtsynthesis was bserved at lw light intensities, which relatinship is independent f temperature. At high light intensities (light saturatin) the rate f assimilatin f carbn dixide des nt increase with an increase in light intensity. In this range, hwever, the increase is dependent n temperature. The transitin frm light limitatin t light

29 57(7) 23 -<-» c * u c^i <-> c u u C C a * C r<\v< i -H-H-H-^-H m r- O ON VO - ^t r* r* ON f^vovovovovo i -H-H-H-H-H-W ON «N VO J^. IO ~ cs IOJ^OCON E as *>^ T3 >> J3 O S"A ft.- + O OO Tf VO O Tf ^ ON in TT <Nr^ Tf rvi 4 *-*» q»n(no in O O in n ^ ir> ^r fs * ) n N 1 t» C c3 as u. U 3 c IT) >> T -1 w*4 ON 6 u ft C i «'t "t Tf w i^ i -H -f I-H-H-H -f 1 -H r- v ON r^- m ^n<h\\ r<«w(n^m«i -H-H-H-H-H-H-N ^ M ^ n r ^ ^ «^nv\ «-N rf TJ-n > i -H-H-H-H -H-H ^ m r- r^ r- rsi v rf * *»-i «S Tt \ f"- m Tf v «^t rf i -H-W-H-H-H+i ^- <N «n m WN OS ON - h 'S P C6 >> * 8 g "a s 's.s «$ ei^ p r r^ <s»-^ t^moo^on- CN «tn ON Tf f- '^fsj (S ~ p O q <OrcsNOv^«nm r^ *-<»-* «-^»n v TJ- Tf rn O ^ <S O «ON ^t fn «fn <N <N C ^ P3 * s fcj GO Urn m <N ft -< O :2 ^-< CI (N r <N fvj i-^ i -ti-u -H-H-H-H-H ^H m ' i ^ r ON rt - t^ m v t^- ON <N ^ <S CM ^ <S ^ i -H-H-^-H-H-H-^ O *^ t^ Tf OO rf ^ ^ M i O t N O N n < rf«t r«n (S (S i -H-H-H-H-H-H-H Tf OM^ OO (S ON >n ~ ^ cs rf v t c*> rt SOVO rt rf i -H-fi-fm-H-fi O^t^ON -^ CS»n t^ ON ON <~ c x 8 J2 E *. I^ vo cs O OOON r-»-«^vh» ^ s cv ON m m(m M ^ OOOOOvt^ m r n «^ M T t N M n n w N t S w p «^ i ^ O M I O O O n M O f^fncsts-h^ O m O «n O v vd v v m «m rs f^nts«*i C 5 >» phts saturat «*-i ti O -C in rlig. -, ^5 'O { «?? Ctf Jg **-3 > Cj ^j i^; S c c t»» U) 1 1 1» ^» *» XX -*f «v t-* *r> v X T t t^f^a^vl r i ^ri f^: a^' r i r^^f^on^fnifn^t^on^ C4 «H 1 1 ^^ X ^^ X ON > O M I ^ V-> r <AJ «(N m «IO Or4rxt-^r4r^Tt l 1 1 <g> ^5 ^5 *-* i < * «XX f> *-^ r T} - Ci»-N c O < \ ^ ri <n \ <- X 1 1. X X ON» Tf OO <S VO e inn es, ui > W J ^Q 13 *-* a HH u <D C JZ 3 *- <1> C 3 w C D, c E CJ ">> X O JC

30 24 57(7) saturatin depends n the density f the suspensin. Therefre, in ur experimentsalways the same density was used. In figure 12a the rate f phtsynthesis against light intensity is represented at 25 C and 17.5 C. Saturatin ccurs at abut 38, ergs/cm 2 sec. at 25 C, and at 25, ergs/cm 2 sec. at 17.5 C. The figure is very similar t thse btained by ther investigatrs. S far, we have studied the inhibitin f phtsynthesis by pisns nly at light saturatin. It seems imprtant t knw, therefre, whether the inhibitin under light limitatin is the same as that under light saturatin, r whether it is smaller. The latter situatin wuld be likely t arise in case f specific interactins f the pisn with definite catalists. In figure 12b, which represents ne ut f 5 experiments, the inhibitin is given at different light intensities, in three cncentratins f CaDz, viz., 1.2 X 1 5 ; 5.2 X 1-6 ;3. X 1 6 M at 25 C. The crrespnding figure 12cshws the inhibitin at 17.5 C with 3. X 1 6 and 1.18 X 1 6 M. It is clear, that at bth temperatures rughly the same inhibitin ccurs at light limitatin and at light saturatin. Hwever, the inhibitin is much mre prnunced at 17.5 C than at 25 C,s that lwer cncentratins f CaD2had t be used t btain the same percentage f inhibitin. This cnfirms the bservatin reprted in the preceding sectin. In table VI a and b, the inhibitin percentage, btained in these experiments, is calculated fr light limitatin and light saturatin. The percentages f inhibitin in the light limiting range and in the light saturatin range are the same within the experimental errr. mm 2 / hu x1 4 ergs/cm 2 sec FIG. 12a. The rate f phtsynthesis f Chlrella in WARBURG buffer n. 9 versus light intensity, at 25 C ( ) and 17.5 C C (+ +); ne ut f 5experiments.

31 57(7) 25 mm' 2 /hu _ /? x1 ergs/cm sec. FIG. 12b. The rate f phtsynthesis f Chlrella in WARBURG buffer n. 9 during the first hur after additin f CaD 2, versus light intensity, at 25 C C ( = cntrl, - 3. x 1" 6 M, + + = 5.2xl~ 6 M,and a = 1.2 x 1-5 MCaD 2 ). mm 3 2 / hu 4 r D x1 ergs/cm 2 sec. FIG. 12C. The rate f phtsynthesis f Chlrella in WARBURG buffer n. 9 during the first hur after additin f CaD 2, versus light intensity, at 17.5 C C ( = cntrl, + + = 1.18 x 1~ 6 M, and = 3. x 1~ 6 M CaD 2.

32 26 57(7) TABLE Via. The inhibitin f phtsynthesis in Chlrella during the first hur after additin f the inhibitr at light intensities f 2, and 55, ergs/cm 2 sec., at 4 cncentratins f CaD 2, at 25 C, as an average f 5 experiments. Cncentratin f CaD 2 (1' 6 M) Rate f phtsynthesis in mm 3 /hr. 2, ergs/cm 2 sec. Inhibitin in per cent. Rate f phtsynthesis in mm 3 /hr. 55, ergs/cm 2 sec. Inhibitin in per cent ± 1 41 ±2 57 ± 1 7 ± ± 1 42 ±3 59 i TABLE VIb. The inhibitin f phtsynthesis in Chlrella during thefirst hur after additin f the inhibitr at light intensities f 1, and 55, ergs/cm 2 sec, at 5 cncentratins f CaD 2, at 17.5 C C. Cncentratin f CaD 2 (1-«M) Rate f phtsynthesis in mm 3 /hr. 1, ergs/cm 2 sec. Inhibitin in per cent. Rate f phtsynthesis in mm 3 /hr. 55, ergs/cm 2 sec. Inhibitin in per cent The fact that the inhibitin is the same at light saturatin and under light limitatin may lead t the suppsitin that there is a narctic actin f CaD2 n phtsynthesis. Narctic pisning is generally less specific than enzymatic pisning, bth with regard t the mlecular structure f the pisn and t the cnstitutin f the catalytic systems affected by it. Hwever, the very lw cncentratin f CaD2 (1 6 t 1 5 M) in which inhibitin ccurs, is likely t be due t specific interactins withdefinite catalists rather than t an indifferent mechanism f surface blcking. In additin, the irreversible inhibitin f phtsynthesis with CaD2 (fig. 8) seems t cntradict the assumptin f a narctic pisning. The cnclusin seems t lay at hand that CaD2 interferes with a type f enzyme present in lw cncentratins. It is pssible, hwever, that in ur experiments the expsure t CaD2was t lng and the ph t high, which may have led t irreversible injury. GAFFRON (23) fund phtreductin in hydrgen adapted algae less sensitive twards hydrxyl amine than phtsynthesis. He cncluded that hydrxyl amine nly participates in the xygen liberating reactin f phtsynthesis, in which it acts as an enzyme inhibitr. WELLER and FRANCK (8), hwever, fund that the inhibitin f phtsynthesis by hydrxyl amine was independent f light intensity. It is evident that in ur experiments CaD2 yields curves similar t thse WELLER and FRANCK (8) btained with hydrxyl amine. In this cnnectin it may be remarked that LIPSCHITZ (37) bserved that armatic nitr cmpunds in bld are reduced t hydrxyl amines, and then t nntxic prducts. The first reductin, hwever, prceeds faster than the secnd, s that accumulatin f hydrxyl amine may ccur. It is pssible that the

33 57(7) 27 mm 2 /hur 3 r- 2 - t x 1 4 ergs/ cm 6 sec. FIG. 13. The inhibitin f phtsynthesis f Chlrella with hydrxyl amine versus light intensity, at 25 C, in WARBURG buffer n. 9 ( = cntrl, -\ + =.95 x 1-*iM, x x x 1" 4 M, = 1.4 X 1" 4 M NH 4 OH.HCl). inhibitin f phtsynthesis with CaD2 prceeds thrugh a reductin t a hydrxyl amine like cmpund. It seemed imprtant, therefre, t repeat the experiments f WELLER and FRANCK (8) with hydrxyl amine and als t measure the inhibitin f phtreductin with CaD2 (fr the latter see Chapter V). In figure 13 the inhibitin f phtsynthesis with hydrxyl amine at different light intensities at 25 C is represented in ne ut f 7 experiments with similar results. We thus cannt cnfirm the results f WELLER and FRANCK (8). Thugh in ur experiments lwer cncentratins f hydrxyl amine (.95, 1.16 and 1.4 X 1 4 M) have been used than thse applied by WELLER and FRANCK (2.5 X 1 4 M), ur results suggest that even in higher cncentratins, inhibitin is much strnger in the light saturatin range than in the light limiting range. 6. DISCUSSION A linear relatinship between the percentage f inhibitin and the cncentratin fr varius inhibitrs (n a lgarithmic scale) was bserved between 2-8% inhibitin at light saturatin (figs. 7a, 7c, 7d, 7e, 7f). Beynd the mentined degrees f inhibitin the curves apprach threshld and cmplete inhibitin mre r less asympttically. Insme cases, therefre, nly the cncentratin causing 5% inhibitin was given. With ethyl urethane (fig. 7b), hwever, a bend in the curve ccurred at.1-.2 M, which was ascribed t a desiccating effect by the smtic value f the inhibitr at these high cncentratins (p. 16). A relatinship between the inhibitin and the cncentratin f the inhibitr was derived by TAMIYA (68) and rendered in the frmula: G n H = n_ _Qn

34 28 57(7) in which H G n = degree f inhibitin (per cent inhibitin in ur experiments, divided by 1). the cncentratin f the inhibitr. a cnstant, crrespnding t the cncentratin causing 5% inhibitin. a cnstant, representing the rder f inhibitry actin. A simpler frmula can be derived frm that given abve, viz. lg G = l H H /n lg + lg. By representing lg G versus lg straight curves will 1 H l ti result frm which the slpe n and the intercept, can be calculated. Frm these cnstants inhibitin curves were calculated and drawn in the figs. 7a, 7b, 7c, 7d, 7e, 7f. The calculated curves fit the bserved values well, which emphasizes the validity f the frmula. The value n is characteristic fr each inhibitr, and as a rule remains cnstant underdifferent cnditins, e.g., with CaD2 at different temperatures. The value (5% inhibitin) may vary, e.g., with temperature, ph (Chapter IV) and light intensity. The results represented here cnfirm thse btained by TAMIYA (68). Hwever, with hydrxyl amine ndefinite decrease in inhibitin was bserved at lwer temperatures (table V). His experiments, hwever, were carried ut at 25 and 4 C, and at a lwer ph f the medium. TABLE VII. Cmputed values f n (rder f inhibitry reactin) and (cncentratin in Mgiving 5% inhibitin) fr varius inhibitrs with respect t phtsynthesis in Chlrella. Inhibitr n I n II n III n IV CaD 2 ethyl urethane phenyl urethane hydrxyl amine 2,4-dinitrphenl sdium azide 1 I = ur experiments IV = WARBURG (71) 4.2 x 1~ x x 1" x 1" x 1~ x 1" x 1" x x x 1" /2. x 1" 6 (6cc) 13.7 x 1" 6 (3cc) 6.5 x 1-6 II = TAMIYA (68) III = WINTERMANS (83) x 1" 1 5. x 1" 4 In table VII cmputed values fr n and are represented fr varius inhibitrs, als frm experiments carried ut by ther authrs (TAMIYA [68], WARBURG [71], WINTERMANS [83]). There is a general agreement by these authrs in the value f (5% inhibitin) with the same inhibitr, as may be seen frm the table. It is again clear that an exceptinally lw cncentratin f CaD2 already inhibits phtsynthesis. Only the bservatins f WINTERMANS (83), cncerning inhibitin with 2,4-dinitrphenl (at ph 4 t 5) are in the same cncentratin range as thse with CaD2. In additin t this, HOLZER (3) demnstrated a far smaller inhibitin ( M being required) with 2,4- dinitrphenl (at ph 8 t 9), which agrees with ur results (at ph 9.2). This increase in inhibitin with a decrease in the ph, which als may ccur with sdium azide, has been discussed by WINTERMANS (83) and by SIMON and BEEVERS (61,62). The cmputed rder f the inhibitry reactin n was the same in these cases.

35 57 (7) 29 Thugh in ur experiments the value fr with hydrxyl amine agrees with that btained by TAMIYA (68), a different rder f the inhibitry reactin was bserved. It is nt yet understd, why in ur case an rder f 3 was fund. This hardly can have a real significance, s that, prbably, the validity f the frmula under discussin, is nt sufficiently general t be applied t all srts f inhibitrs. In cnnectin herewith, als the different behaviur in inhibitin f phtsynthesis at light limitatin in ur experiments and in thse f WELLER and FRANCK (8) is nt clear. Pssibly, the frmula applied by TAMIYA (68)is nt applicable t all inhibitrs, whilst preparatin f the cultures certainly affects the results, as was already mentined in the intrductin f this Chapter. A large increase in inhibitin f phtsynthesis with decrease f temperature was fund nly with CaD*. T btain the same inhibitin percentage, lwer cncentratins had t be used at lwer temperatures, which amunted rughly t a Q 1 (cne. [T + 1] C/cnc. T fr 5 per cent inhibitin) f 5 (fig.11). It is nt yet understd, why such a high Q 1 ccurs. This fact is pssibly nt due t higher uptake f CaD-2, since at all temperatures a cnstant inhibitin was attained after the same time interval elapsed since the additin f the inhibitr. CHAPTER IV INHIBITION OF PHOTOSYNTHESIS WITH CaD 2 AS COMPARED WITH THE INHIBITION OF OTHER METABOLIC PROCESSES IN CHLORELLA 1. INTRODUCTION In the preceding chapter we have cmpared the influence f CaD-2 n Chlrella phtsynthesis with that f ther inhibitrs. Under cmplete inhibitin f phtsynthesis by CaD-i we have ften bserved negative manmeter readings which even excelled thse f the dark respiratin f the nn-inhibited cntrl (Ch. III). In cnnectin herewith we have studied respiratin in the presence and absence f glucse with different cncentratins f CaD*. These experiments will be discussed in this chapter. Mrever, the inhibitin f phtsynthesis with CaD 2 was investigated at different ph f the medium and in flashing light. In additin flurescence measurements were made, Finally, experiments regarding the inhibitin by CaD 2 f auttrphic and hetertrphic grwth f Chlrella in lng term experiments will be presented. 2. INHIBITION OFENDOGENOUS AND GLUCOSE-STIMULATEDRESPIRATION Respiratin was als measured in the WARBURG apparatus. Chlrella was suspended in distilled water, and.2 cc 1 N KOH was pipetted int ne f the side arms f the vessel, which was sufficient t absrb all carbn dixide prduced in respiratin. Cnsequently, the negative change in the manmetric reading is due t xygen uptake. Smetimes, when very small readings culd be expected, the suspensin was made up t a ttal quantity f 3 mm 3 f

36 3 57 (7) cells per vessel. Fr reasns f cmparisn these results have been cnverted t 2 mm 3, the amunt nrmally used. In the experiments with the glucsestimulated respiratin the cells were washed and suspended in distilled water with.5 % glucse. This quantity f glucse was sufficient t btain a cnstant rate f respiratin fr at least 4 hurs. Experiments n endgenus respiratin Measurements were made in a range frm t 1 x 1 3 M CaD2,fr 2 hurs after additin f the inhibitr. The influence f CaD2 n respiratin is shwn in figure 14 as an average f eight experiments at 25 C. The relatively high mean errr in these experiments results frm the small readings, the rate f respiratin being lw (rate f the cntrl 15 mm 3 2/hur). In ur experiments, hwever, the respiratin f the cntrl never exceeded that f cells with CaD2. On the cntrary, there is a stimulatin f respiratin which increases with increase f the CaD2 cncentratin up t abut 2.2 X 1 4 M. This high rate f respiratin was maintainedeven at 1 X1 3 M CaD2. The ultimate stimulatin at this cncentratin ranged frm 35 t 1% and remains at that level fr at least three hurs. The reversibility f this stimulatin has nt been tested by washing experiments. Experiments n glucse-stimulated respiratin Because f the lw rate f respiratin, it may be assumed that nt all active places n the enzyme systems are ccupied, which prbably explains the relative insensitivity t CaD2. By adding.5 % glucse t the Chlrella suspensin, the rate f respiratin is increased abut 4-8 fld. The same range f cncentratins and the same prcedure f measurement have been used as fr endgenus respiratin. Usually, glucse was added ne hur befre the experiments started in rder t avid interference f the adaptatin frm endgenus t glucse respiratin, which takes place during the first hur. After that, a cnstant rate was bserved fr at least 4 hurs. The inhibitin f glucse-stimulated respiratin is shwn in figure 15 as an average f 17 experiments at 25 C. The inhibitin increases steadily with the increase f the CaD2 cncentratin, but the xygen uptake still was45 % f the cntrl at the highest cncentratin. Frm the curse f the curve a further increase in inhibitin may nt be expected at higher cncentratins. This, hwever, hasnt been tested, since 1 x 1 3 M CaD2was thehighest cncentratin available. At this cncentratin, the rate f glucse respiratin was still higher than the stimulated endgenus respiratin at the same cncentratin f the pisn (see previus sectin). Washing experiments were perfrmed at 5 cncentratin levels. After a 2 hurs treatment with CaD2, Chlrella was centrifuged, washed three times and measured again after being suspended in.5% glucse. It is clear that the inhibiting effect n respiratin is at least partly reversible, as may be seen frm figure 15. This reversibility cntradicts the results btained n phtsynthesis. Anther difference is that, unlike in phtsynthesis, the inhibitin in the glucse respiratin remains cnstant at any cncentratin. The inhibitin reaches a cnstant value within 2 minutes, as may be seen frm figure 16. This fact will be discussed latern in this paper (Chapter VI).

37 57(7) 31 O L. _ Q> C E ' a D 3 Pi < J3 E J - Q O O "l O I O C3 T3 a c. CO in I -H O c 'a CO CO CD O C ) DO O T3 C u CO t c3 cn c u f I I 1 I I I L X 1 C\J x: H CO CO >

38 32 57(7) % inhibitin M Ca D 2 FIG. 15. The inhibitin f the glucse-stimulated respiratin at different cncentratins f CaD 2 ( ) and theeffect f washing n the inhibitin (H h) at25 C (Ch/rella suspended in.5% glucse slutin with.2cc 1 N KOH in the side arm f the WARBURG vessel). % inhibitin 1 r ^. *» m m 4 / ^ f y! hurs after additin fcad 2 FIG. 16. The inhibitin f the glucse-stimulated respiratin f Chlrella versus time after additin f the inhibitr at 3 cncentratins f CaD 2 ( -= 5.2 x 1~ 4 M, x 1" 4 M, x 1-5 M CaD 2 ).

39 57(7) INHIBITION OF PHOTOSYNTHESIS AT DIFFERENT ph VALUES OF THE MEDIUM S far, measurements have been made in WARBURG buffer n. 9, cnsequently the inhibitin f CaD2 has always been measured as a change in O2 (Methd I). In rder tfind ut whether the uptake f CO2was inhibited t the same degree, the phtsynthetic qutient p = -O2/CO2 was calculated by measuring the inhibitin at ph 5.8 and ph 7.2, using the tw vessel methd. Frm the tw manmetric readings, the uptake f CO2 and the release f O2 can be cmputed as already described (p. 9, Methd II). The same quantity f cells was suspended in 2 and 5 cc phsphate buffer respectively. The measurementswere made atlight saturatin, at25 C. The phtsynthetic qutient was determined withut CaD2 and with 1.56 X 1 6 M CaD 2. The results are shwn in table VIII. TABLE VIII. The phtsynthetic qutient f Chlrella at ph 5.8 and ph 7.2 (sdium phsphate buffer) with and withut additin f CaD 2. Each qutient representing the average f 5 experiments at 25 C C. ph Average Cntrl Extreme values 1.56 x 1~ 6 MCaD 2 Average Extreme values ±.1 ( ) ( ) 1.5 ± ±.1 (1.-1.1) ( ) Within the limits f errr, n differences in the phtsynthetic qutient ccur between the cntrl and the suspensin cntaining 1.56 X 1 6 M CaD2, neither at ph 5.8 nr at ph 7.2. The phtsynthetic qutients in the cntrl and in the presence f CaD2 at ph 7.2 are in general smewhat higher than thse at ph 5.8. The differences, hwever, are nt significant. As there was n effect at all n the phtsynthetic qutient (p), the inhibitin f phtsynthesis at different ph values has been measured in phsphate buffers using the ne vessel methd (Methd III) in rder t enable a wider variety f treatments. The cmputatins in Chapter II shw that the influence f errneus p-valuesbecmes smaller with larger vlumes f fluid (lwer Vc values). Als the resulting manmetric readings are higher, which is anther advantage f using as much fluid as pssible. In ur measurements, therefre,weused 5 cc, in which disturbing effects due t mixing f the fluid in the main part f the vessel and in the side arm d nt yet ccur. The influence f ph n the inhibitin f phtsynthesis by CaD2 is given in table IX. The measurements have been made in phsphate buffers (ph 5.9 and 7.2) r in carbnate bicarbnate buffers (ph 9.2 and 1.4). In the latter case the calculatins prceed accrding t Methd I (Chapter II). It was mentined in Chapter II that cnsiderable errrs in the cmputed rate f gas exchange ccur if n crrectin fr CO2 retentin is applied. Since this nly affects the verall vessel cnstant, it has n influence if the inhibitin is represented as a percentage f the cntrl (see clumns 3 and 5). N ph effect was bserved inthe phsphate buffers (clumns 3 and 7). In the carbnate bicarbnate buffers the inhibitin at ph 9.2 was less than at ph 1.4. It is pssible, hwever, that in the latter case an unfavurable effect f the high ph ccurs, which was als fund in cntrl experiments (Chapter II). The cncentratins f CaD2 yielding 5 % inhibitin were nt entirely the same

40 34 57(7) <N ON X, * C/5 t-.4) fej 3 X> O * - ^ «PC a </) JC ex c "* <N r^ T3 C 3 ti I c.2 E c.2 A* US CO -fh-l O Tf r^ 1 1 X CO t I r r Tf r fs O H-H-H-H-^x f- r rf <N r ^, CJ rj OS «K N a E 3 * * "O <u E JS rf v_ X a.< > c <u u-.w fes mm - +-» c3 *i c3 u U «>><N -*- C3 #* x> 5 ^^^ ^ J**, ^! U. c * #C/5 *w5 CL> XI «* c >^ t/5 ' O ' 4 > XJ a C M «H 2 X)' v-«rf*l. ' a -C H 'w? u-,4> te 3 «< C3 C x> s- cs X C3 C OX) c a c X 3 <,p * Tf T3 c c3 CM CM X,2 S; C *^ C/l I- c.2 c P *J «5 /- I u (-1 /^.2 ~ c.2 I I C O.2 - c:~ s a e V >» - W (/J _ s c 2.2 h cs s ^. <- r " CM O «n m v -^ O <N «TJ- rs) vo ON VO r4 N r ^ H-H-W-H-H-H-Hx Tfr^vccTf,^ -i (S m v \ " Tf»r4 vi/~><n ««OOOCN V ) Tf Tf r m rs t I ^frc>tf>i/~>r-- -H-H41 -H-H-H -U x CN Tt v r- ^ u->rnmr- (SOMOOOCAiOO TfrOTtOO«OV->r»cO -H-+]-H-H-H-H-W y vrnmmm O N O M O i - O O VOM ONOOOO«^«lc (N "OQ\» (J\ n ON C «<N>VD ".2 O X) <-"* "Hx- O.E rs n CM C u E C/3 Z O *ZJ c a a

41 57(7) 35 7 r- Efrel. units) Q 1 FIG. 17. The extinctin f CaD 2 measured in relative units versus ph (buffers f different ph, measurements with 5.8 x 1~ 6 M CaD 2 ). in phsphate buffers and in carbnate buffers. Hwever, the experiments were made n different data, which may, at least in part, accunt fr this difference. The rder f the inhibitry reactin was 2 in all cases, calculated as indicated n p. 28. If nly undissciated mlecules penetrate int the cell, a decrease in ph will increase the frmatin f HD mlecules. As nly thed-in is intensely clured, the variatin in extinctin f a slutin f CaD2 with ph mayfurnish a measure f the dissciatin. In figure 17, the extinctin, n a lgarithmic scale, is pltted against ph fr 5.8 X 1 6 M CaD2, giving abut 75% inhibitin f phtsynthesis at ph 9.2. Frm this figure it is clear that a decrease in extinctin nly ccurs at ph values lwer than 4.. It is prbable, therefre, that strnger inhibitin f phtsynthesis is nly perceptible belw this value. Hwever, measurements f the rate f phtsynthesis at ph values belw ph 4. are unreliable under these nn-physilgical cnditins. The internal ph f the cell, n the ther hand, is t a high degree independent f the ph f the utside medium. PH 4. THE INHIBITION OF PHOTOSYNTHESIS IN CONNECTION WITH CHLOROPHYLL FLUORESCENCE Excited chlrphyll may dissipate energy int flurescence, heat r as ptential energy t anther mlecule during cllisin. Only asmall fractin f the absrbed quanta is dissipated as flurescence. Hwever, the intensity f flurescence is a direct measure f the number f excited chlrphyll mlecules present. Inhibitrs changing the initial slpe in phtsynthesis will usually affect flurescence, whereas thse nly influencing the saturatin level will have n effect n flurescence r affect it nly indirectly. Measurements have been reprted by KAUTSKY and HIRSCH (32), KAUTSKY and SPOHN (33), and laternby FRANCK etal. (17) with Chlrella, and by WASSINK etal. (75, 76, 77) with Chlrella, Diatms and Chrmatium. Flurescence measurements were made in small rectangular vessels (3 X 2 X 1 cm). A high pressure mercury vapur lamp was used as a light surce in these

42 36 57(7) experiments, because the relatively small amunt f red light culd easily be remved with a 6 cm half saturated cpper sulphate slutin remving all the infra-red, red and range cmpnents f the light. Since flurescence directly depends n the incident light intensity, the mercury lamp was fed via an a.c. stabilizer. Weused the same equipment as SPRUIT and WASSINK (65), measuring the flurescence light with a caesium pht-electric cell cnnected with an a.c. amplifier. The experiments were perfrmed at rm temperature (abut 19 C).Different light intensities were btained by using cpper wire screens f different mess width. In figure 18, the flurescence is represented in relative units at different light intensities and at different CaD2 cncentratins. There is a linear relatin up t a light intensity f X 1 4 ergs/cm 2 sec. after which the slpe increases. The transitin range agrees clsely with the reach f light saturatin in phtsynthesis (fig. 12a). With 1. X 1 6 M giving nearly cmplete inhibitin f phtsynthesis, the flurescence is smaller, bth at high and lw light intensities. At still highercad<2 cncentratins (2.6 X 1 5 M) a further decrease in flurescence was bserved, especially at high light intensities, resulting in straightening the curve. Atlwer cncentratins fcad-2, giving5 % inhibitin f phtsynthesis, a decrease in flurescence was als bserved. rel. units 4 r x1 ergs/cm 2 sec. FIG. 18. The effect f CaD 2 n flurescence f Chlrella versus light intensity at different cncentratins ( = cntrl, x x = 5.2 x 1~ 6 M, = 1 X 1 _5 M, + + = 2.6 x 1~ 5 M CaD 2 ).

43 57 (7) 37 The results indicate that CaD2 accepts the energy transferred frm the excited chlrphyll and des s even with a higher prbability than the nrmal energy acceptr, since flurescence is decreased. It is knwn that nitrphenls generally accept energy quite easily, withwhich ur results arein accrdance. 5. INHIBITION OF PHOTOSYNTHESIS WITH CaD2 IN FLASHING LIGHT Intermittent light was already applied in the study f phtsynthesis by BROWN and ESCOMBE (1), and later by WARBURG (71). In the classical studies f EMERSON and ARNOLD (13), imprved methds fr the study f flash light were used. By variatin f the dark perid, they fund that, after the additin f cyanide, causing 6 % inhibitin in cntinuus light, the yield f phtsynthesis per flash increases with increasing dark perid and was the same as the cntrl if the dark perid was sufficiently lng. The inhibitin f phtsynthesis with phenyl urethane, n the ther hand, was nt cunterbalanced by a lnger dark perid. Latern, inhibitin experiments f phtsynthesis in cnnectin with flashlightwere made by WELLER and FRANCK (8) and TAMIYA (68). Our experiments were perfrmed at 3 C, with Chlrella suspended in.2 M WARBURG buffer n. 1 (ph 8.7). CaD2was added t the suspensin befre the latter was pipetted int the reactin vessel. The yield f phtsynthesis was measured with a recrding vlumeter, the design f which has been described in full detail by KOK (35). Different light intensities were btained by using cpper screens f different mess width. By varying the width f the slit in the rtating disc and the rtatin speed, different dark perids (.5-.8 sec.) culd be cmbined with the same flash time (.32 sec). With the aid f this apparatus light intensity curves culd be made in cntinuus light as well as in flash light. In the intervals between the flash light partsf the experiment, dark peridswere intercalated in which respiratin has been measured. In these experiments an increased respiratin up t 6 % was bserved in the dark with increasing CaD2 cncentratins. The high respiratin rate may giverise t surces f errr in the cmputatin f the yield f phtsynthesis especially in case f large dark intervals between the separate flashes. Especially s, because the pssibility f an influence f the flashes upn the respiratin in the dark intervals cannt be excluded a priri. The results btained in cntinuus light cnfirm thse btained with the WARBURG apparatus (figs. 12b and 12c) and are nt recrded here again. In figure 19, the per cent inhibitin at saturatin is pltted against the length f the dark perid at different cncentratins f CaD2. It is clear that neither at 6.9 X 1 6 M CaD 2 nr at 4.7 X 1 6 M CaD 2 a decrease in the inhibitin f phtsynthesis is bserved even with dark perids f.8 sec. These results are very similar t thse btained with phenyl urethane in flashing light experiments (13). 6. INHIBITION OF AUTOTROPHIC AND HETEROTROPHIC GROWTH The grwth experiments were perfrmed in 3 cc erlenmeyer flasks cntaining 15cc f WARBURG culture medium. The flasks were inculated with the same amunt f cells i.e. 15 mm 3 cells/flask and then placed n the shaking device. By interchanging the places f the erlenmeyers every day, differences in light intensity n the shaking device were eliminated.

44 38 57(7) % inhibitin 1 r i dark time in secnds.8 FIG. 19. Per cent inhibitin f phtsynthesis f Chlrella, in cntinuus and in flashing light versus length f dark perid at different CaD> cncentratins (H + = 4.7 x 1~ 6 M, and = 6.9 x 1" 6 M CaD 2 ). Grwth has been measured by centrifuging a fixed vlume in a TROMMSDORFF tube fr 11 minutes at a speed f 225 revlutins per minute. The height f the clumn in the capillary f the TROMMSDORFF tube has been taken as a measure f the density f the culture. The uptake f CaD 2 in the grwing culture was determined by measuring the transmissin f the supernatant in the TROMMSDORFF tube at 48fi in a pht-electric clrimeter. The cncentratin f CaD 2 culd be read frm a calibratin graph. It smetimes happened that the cultures became turbid, and the supernatant did nt becme clear even after centrifuging fr a lng perid f time. This tends t suggest a higher cncentratin f CaD 2 than is actually present. The turbidity is prbably due t calcium cmpunds in clldial suspensin. Assuming that the turbidity ccurs in the same way in a culture withut CaD 2, ne can take the supernatant f the cntrl instead f distilled water fr cmparisn, as has been dne in the first experiments. Hwever, as grwth in these cntrls is strnger than in cultures with CaD 2, higher turbidities ften ccur in the cntrls, resulting in t lw estimatins f the CaD 2 cncentratin. Better results were btained by diluting the supernatant with distilled water and shaking it with a small vlume f methyl-isbutyl ketne. The ketne phase cllects at the tp and cntains all CaD 2, whereas mst f the turbidity remains in the water phase. Auttrphic grwth Each grwth experiment cnsisted f a range f 8 erlenmeyer flasks with CaD>2 cncentratins frm t 6.6 X 1 6 M. The cncentratins f CaD-2 were measured directly after inculatin. After 5 days, the grwth f Chlrella and the cncentratin f CaD2 were measured. In table X, the number f cells is given after 5 days as an average f 6 experiments (clumn 2). The cncentratin f CaD2 after inculatin, the inhibitin f grwth and the cncentratin f CaD2 at theend f theexperiments areshwn in clumns 1,3, and 4respectively. The inhibitin f grwth increases with the increase in the cncentratin f CaD2 (clumn 3). Cmparing these results with thse n phtsynthesis (figure 7a,25 C) it may be seen that the range f inhibitin is abut the same. The absrptin f CaE>2 tends t increase with the cncentratin (clumn 5);

45 57(7) 39 TABLE X. The inhibitin f auttrphic grwthin Chlrelta withdifferent cncentratinsf CaD 2 asanaverage f 6experiments. 1. Cncentratin f CaD 2 (1-«M) directly after inculatin 2. Quantity f cells in mm 3 /1 cc after 5 days *) Xumer. value 2 ) 3. Rel. grwth rate 2 ) Per cent inhibitin 4. Cncentratin fcad, (1"*M) in supernatant after 5 days 5. Absrptin f CaD, (1~ 8 M) frm 1 cc suspensin by the cells in 5 days.66 ± ± ± ± ± ± ±.6 4. ± ± ± 6 3. ± ± 2 1. ± ± ± i ± ± ± ± ± ) Initial density 1 mm 3 cells in 1 cc. 2 ) Relative grwth rate: lg. increase f cell density per day. this is even mre s per cell, because f the strnger inhibitin f grwth. We have investigated whether the decrease in cncentratin f CaD2 might be merely due t the presence f ptassium in the medium. That this culd nt be the explanatin was demnstrated by the bservatins that n decrease in cncentratin ccurred at 8.8 X 1 6 M CaD2in erlenmeyer flasks with water r a WARBURG medium withut algae, neither in dark nr in light, after 5 days. Hetertrphic grwth Each experiment cnsisted f a range f cncentratins frm t 1.73 X 1 5 M CaE>2. The inculatin and the measurements have been made in the same way as already described abve in the sectin n auttrphic grwth. These experiments were perfrmed in the light (35-45 lux),in the sameway as thse n auttrphic grwth, 15 gr. glucse/1. having been added t the medium. The results are represented in table XI. TABLE XL The inhibitin f hetertrphic grwth in Chlrella with different cncentratins f CaD 2 as an average f 5 experiments. Cncentratin f CaD 2 (1 6 M) directly after inculatin Quantity f cells in mm 3 /1cc after 5 days 1 ) Numerical 2 ) value Rel. grwth rate 2 ) Per cent inhibitin 1.53 ± ± ± ± ± ± ± ±.6 1 ± 1 12 ± 2 1 ± 1 12 ± 1 1 ± 1 7 ± 25 4 ± i x ) Initial density 1 mm 3 cells in 1 cc. 2 ) Relative grwth rate = lg. increase f cell density per day.

46 4 57 (7) N definite measurements f CaD2 culd be made at the end f these experiments, because f the high turbidity f the glucse cultures. The extractin methd f D with methyl-isbutyl ketne was nt fllwed, since it was nt yet knwn t us during these experiments. Visually, the uptake f CaD2 was higher than in the auttrphic series wing t the higher CaD-2 cncentratins, and the higher density f the suspensin, which was 4. and 1. mm 3 in the cntrl f the auttrphic and hetertrphic grwth series respectively. It may be seen frm tables X and XI that the inhibitin f hetertrphic grwthis smaller. The cncentratin at which cmplete inhibitin f auttrphic grwth ccurs hardly reaches the threshld fr inhibitin f hetertrphic grwth. 7. DISCUSSION Generally, inhibitin f respiratin is fund at higher cncentratins f the inhibitr than that f phtsynthesis. Up t the present the reverse has nly been reprted fr cyanide inhibitin in Scenedesmus (GAFFRON [21 ]). Armatic nitr-cmpunds usually increase the rate f respiratin t a high extent in cncentratins in which synthetic prcesses are decreased r cmpletely inhibited. Inhibitin f respiratin was bserved at higher cncentratins (BEEVERS, [4], KELLY and AVERY [34]). In ur experiments an increase f respiratin frm 3 t 1% was bserved with increasing CaD 2 cncentratins up t 1~ 3 M in the dark. Inhibitin may well ccur at still higher cncentratins, but this culd nt be tested since 1 3 M CaD2 was the highest cncentratin available. The absence f a clear inhibitin f respiratin may well be due t its lw rate. Additin f glucse results in an increase f respiratin up t 6-8 times the value f the endgenus respiratin. An inhibitin up t 6 % f the glucsestimulated respiratin was bserved at 1 3 M. Yet the rate still was nearly 2 times that f the stimulated endgenus respiratin at the same CaD2 cncentratin. It may be seen frm figure 15 that the inhibitin f glucse respiratin tends t increase mre slwly in the regin f 1 3 M. The data cntain n indicatin that the rate f respiratin at still higher cncentratins f CaD2 shuld be belw that f the CaD2 stimulated endgenus respiratin. Hetertrphic grwth was fund t be less sensitive t CaD2 than auttrphic grwth. It may be seen frm table X and figure 7a that in auttrphic grwth and phtsynthesis the threshld cncentratins f CaD2 fr inhibitin are almst the same. Hwever, the cncentratin at which cmplete inhibitin ccurs is higher in phtsynthesis. It must be mentined that the grwth experiments cvered a perid f 5 days, whereas the inhibitin f phtsynthesis was measured fr 3 hurs after additin f CaD2. A prgressive increase in inhibitin f phtsynthesis was bserved (figure 8). It may be cncluded that inhibitin f phtsynthesis and auttrphic grwth present nearly the same characteristics. As threshld and cmplete inhibitin f hetertrphic grwth are much higher, itis prbable that the inhibitin f auttrphic grwth is due t the inhibitin f phtsynthesis, resulting in a shrtage f assimilates, which becmes even wrse by the increase in respiratin. The bservatins f SEEL (58), that nitr-cmpunds in lwer cncentratins are smetimes mre txic than in higher cncentratins, may als be due t a shrtage f assimilates since respiratin increased especially at lwer cncentratins. It is knwn that D is an even strnger inhibitr fr cell divisin

47 57 (7) 41 thanclchicine (GAVAUDAN et ah [24]). Nevertheless, this prcess, still may be less sensitive than phtsynthesis. The bservatins n flurescence and flashing light emphasize the evidence already btained in Chapter III, that CaD2 inhibits equally the energy transferring part and smef the dark chemical parts f the phtsynthetic prcess. Fr, if a pisn inhibits phtsynthesisin the light limiting range, a decrease r an increase f flurescencemay be expected, dependent n whether the pisn has a higher r a lwer energy accepting capacity than the energy acceptr invlved in phtsynthesis. In ur case a decrease in flurescence was bserved with CaD2 in the light limiting as well as in the light saturatin range. This indicates that CaD2 has a high energy accepting capacity. Since it acts, as fund, in lw mlar cncentratins this fact may suggest sme chemical similarity t the nrmal primary energy acceptr in phtsynthesis. If a pisn inhibits phtsynthesis in the light limiting range n decrease in inhibitin percentagewill be bservedin flashing light, cntrary t whatis fund fr a pisn preferably inhibitry in the light saturatin range (13). With CaDi we fund n decreasef inhibitinin flashing light as cmpared with cntinuus light. Thugh these results represent a strng evidence fr a narctic actin f CaD2, the lw cncentratin at which inhibitin already ccurs and the nnreversibility pint t a specific bnd t an enzyme rather than t a nn-specific narctic actin. The results btained at different ph will be discussed with thse in Scenedesmus latern in this paper. CHAPTER V INHIBITION OF PHOTOSYNTHESIS AND GROWTH BY CaD 2 IN VARIOUS PLANT TYPES AS COMPARED WITH CHLORELLA 1. INTRODUCTION S far we have studied the effect f CaD2 n varius metablic prcesses in Chlrella nly. It culd be expected that the range f cncentratins at which grwth and phtsynthesis are affected by CaD2 differs in varius plant species. The inhibitin in ther respects as, e.g., regarding chlrphyll cntent, dry weight, rt length, may differ as well. Strains f Lemna minr\ Lemna gibba, Lemna arrhiza, Scenedesmus, Chrmatium spec. y Nstc species, Rhdspirillum rubrum, Oscillatria spec, and Chlamydmnas are kept in this labratry in sterile stck cultures n agar r in slutin, the latter especially fr Lemna and Oscillatria. The rganisms were transferred repeatedly t fresh agar slants, r fresh culture slutins at shrt intervals in rder t activate the cultures. Hereafter, they were inculated int 3 cc erlenmeyer flasks cntaining 1-15 cc f a nutrient slutin. During cultivatin the nutrient slutin was flushed with a stream f air, enriched with 5 % C 2. The erlenmeyer flasks were placed between flurescent tubes ( day light" type) at a light intensity f 3-5 lux, and incubated at a temperature f 2 ± 2 C. Grwth was measured by centrifugatin in TROMMSDORFF tubes fr varius algae and purple bacteria, by cunting frnds (Lemna) r by visual estimatin f grwth (Oscillatria). Phtsynthesis and respiratin were measured in the WARBURG apparatus. Fr specific details abut culture methds and measurements we refer t the sectins dealing with each species separately.

48 42 57(7) 2. INHIBITION IN Lemna minr', Lemna gibba AND Lemna arrhiza Catin deficiencies in Lemna have been studied frequently, e.g.,byashbyand OXLEY (2), WHITE (82), PIRSON and SEIDEL (46), BIERHUIZEN (5); phsphate deficiency was studied by PIRSON et ah (47) and LINDEMAN (36). T the authr's knwledge, hwever, few experiments were made n the effect f inhibitrs n grwth and phtsynthesis. Details f culturing are as fllws: The slutin wasbased n that f GORHAM(26), cntaining 5 x 1~ 3 ml. Ca (N 3 ) 2, 4 HO; 2 x I" 3 ml. MgS 4> 7H 2 ;5 x 1" 3 ml. KNCV, l x 1~ 3 ml. KH 2 P 4 and.5 gr. ferritartrate per litre with thefllwing additin f micr elements: 2.86 mg. H 3 B 3 ; 1.82 mg. MnCl 2, 4 H 2 ;.22 mg. ZnS 4, 7 H 2 ;.7 mg. MO 3 ;.8 mg. CaS 4, 5 H 2. Unless indicated therwise, 5-1 frnds were inculated per erlenmeyer. Flushing f the cultures with air enriched with 5% C 2 fr three 2-hurs perids per day was sufficient t btain expnential grwth. Grwth was measured daily,by cunting the numbers f frnds. In rder t minimize the effect f different stages f develpment in Lemna minr and Lemna gibba, a yung frnd was rated as i, a medium ne as j, an lder ne as j, and a mature frnd as 1. Frnd area was measured frm phtgraphic cntact prints f the frnds by means f a planimeter. Dry weight was measured by vendrying (at 15 C) 1-2 frnds in small weighing bttles during 2 days. Chlrphyll determinatins were made by extracting the frnds with 9% ethanl. The extractin was repeated three times in a waterbath (5-6 C C) which was sufficient fr ttal extractin. The chlrphyll cncentratin was measured in a BLEEKER clrimeter at 665A. Phtsynthesis was measured in the WARBURG apparatus with 1 t 25 frnds in 5cc tap water per flask (ph 5.5)in the case f Lemna minr and with 1-15 frnds in that f Lemna arrhiza. Theincreaseinfrnd number f Lemna minr, Lemna gibba and Lemna arrhiza is represented in figure 2, in which the frnd number is pltted n a lgarithmic scale. The straight lines indicate a cnstant grwth rate thrughut the experiment, with the highest rate in Lemna minr and the lwest in Lemna arrhiza. frnd number days after inculatin FIG. 2. The increase in frnd number (n a lgarithmic scale) versus time (in days after inculatin) Lemna minr (H h), Lemna gibba ( ), and Lemna arrhiza ( ).

49 57(7) 43 Experiments with Lenma minr and Lemna arrhiza were made at different cncentratins f CaD-2. The results are represented in table Xlla and b as an average f 4 experiments. TABLE Xlla. The effect f different cncentratins f CaD 2 n the rate f grwth, n dry weight, n frnd area, n chlrphyll cntent and n rt length in Lemna minr, as an average f 4 experiments. Cncentratin f CalX (1-«M) Rel. grwth rate ') Numerical value 1 ) Dry weight per 1 frnds mg Per cent tlecrea.se Frnd area mm Per cent decrease Chlrphyll*) cntent per 15 frnds Per cent inhibitin Numerical value 2 ) Per cent decrease Rt length cm Per cent decrease s *) Rel. grwth rate lg. increase in frnd number per day. E -E 2 ) measured as lg. - TABLE Xllb. The effect f different cncentratins f CaD 2 n the rate f grwth and n dry weight in Lemna arrhiza, as an average f 4experiments. Cncentratin f CaD 8 (1-«M) Relative grwth rate l ) Numerical l ) value per cent inhibitin Dry weight per 1 frnds mg Per cent decrease *)Relative grwth rate = lg. increase in frnd number perday. The inhibitin f grwth is represented as a percentage f the cntrl. The table includes als the influence f CaD2 n dry weight, leaf area, chlrphyll cntent and rt length, the inhibitin expressed as percentages f the respective cntrls. Lemna arrhizais the mresensitive ne. Cmplete inhibitin f grwth ccurs already with 4.7 X 1 6 M, whereas in Lemna minr at the same cncentratin the grwth rate still is 66 % f that f the cntrl. Cmplete inhibitin f Lemna minr nly ccurs at cncentratins abve 8.5 X 1 6 M. The same relatin was bserved in dry weight/frnd. The chlrphyll cntent in Lemna minr decreases t abut the same extent as the rate f grwth; leaf area is less affected by additin f CaD> and decreases nly in cncentratins abve 4.7 X 1 6 M CaD2. The number and the length f the rts were reduced enrmusly, with 1.97 X 1 6 M already t abut 2% f the cntrl, and with 4.7 X 1 6 M n rts at all were bserved. These effects are shwn in figure 21which is a cntact print f sme frnds grwn at the different cncentratins (1 is the cntrl, 2 represents 1.97, 3 represents 4.7, and 4 represents 8.5 X 1 6 M CaD 2 ).

50 44 57 (7) 1 z V 1^ ^t FIG. 21. The effect f CaD 2 n rt frmatin f Lemna minr (1 = cntrl, 2 = 1.97 X 1-*M, 3 = 4.7 x 1-«M, and 4 = 8.5 x 1~ 6 M CaD 2 ). The inhibitin f phtsynthesis in Lemna minris represented as per cent f the cntrl, in figure 22, in shrt time experiments (-2 hurs after additin f CaD2) and in lng term experiments (after 24 hurs). The inhibitin in grwth and phtsynthesis in Lemna minr as cmpared with that in Chlrella indicates a slwer uptake f CaD2 in Lemna minr. / inhibitin uu ^ + rs 1 i * 1rs M CaD 2 FIG. 22. The inhibitin f phtsynthesis f Lemna minr, with CaD 2, at 25 C, under light saturatin ( x W ergs/cm 2 sec.), in tapwater; -2 hurs after additin f the inhibitr ( ) and 24 hurs later (H h).

51 57(7) INHIBITIONIN Scenedesmus Extensive inhibitin experiments with Scenedesmus have been made by GAFFRON (23) and NAKAMURA (38). GAFFRON (23) reprted fr the first time a case in which respiratin was suppressed by cyanide withut injury t phtsynthesis. It therefre seemed imprtant t measure respiratin als in cnnectin with CaD2. The rate f respiratin was measured in the dark in a cncentratin range up t 1 X 1 3 M CaD2. N inhibitin at all was bserved at any f these cncentratins. On the cntrary, a stimulatin f respiratin ccurs, which increases with increasing CaD2 cncentratins up t abut 6% at 2.67 X 1 4 M CaD2. This high rate f respiratin was maintained even at 1 X 1~ 3 M. These results are in agreement with thse btained with Chlrella. Grwth experiments were made with Scenedesmus in a WARBURG medium (see Chapter II). The experiments were repeated 3 times with 5 different cncentratins f CaD 2 (,.55,1.9,2.19,3.28 X 1 6 M CaD 2 ). Inhibitin f grwth ccurred already at.55 X 1 6 M (the grwth rate being 75% f that f the cntrl), and cmplete inhibitin was bserved at 2.19 X 1 6 M CaD2. Inhibitin f grwth, therefre, ccurs at smewhat lwer cncentratins than in Chlrella. / Q inhibitin 1 r r6 1» f i i» 11 "5 1 M Ca D 2 FIG. 23. The inhibitin f phtsynthesis f Scenedesmus, with CaD 2 at 25 C, under light saturatin ( x 1 4 ergs/cm 2 sec.), in WARBURG buffer n. 9; 1 ( «)and 3 hurs ( )after additin f the inhibitr. In additin heret sme measurements n phtsynthesis were made at different cncentratins (, 2.63, 5.2, 7.65 x 1 6 M, 1., 1.84, 2.7 X 1 5 M CaD2). In this cncentratin range n inhibitin ccurs in the first hur after additin (figure 23). Inhibitin then became nticeable and increased gradually until after 3 hurs a marked inhibitin was manifest, which furthern remained rather cnstant. These results shw a marked difference with the strng inhibitin f grwth reprted abve, the mre s since the inhibitin f pht-

52 46 57(7) synthesis was less than that in Chlrella. This indicates, that grwth is nt inhibited slely by a shrtage f phtsynthates, as culd be cncluded t in the case f Chlrella in Chapter IV. The grwth experiments were perfrmed in a nutrient slutin at ph 5. t 7. while the rate f phtsynthesis was measured in WARBURG buffer n. 9 at ph 9.2. Therefre, simultaneus measurements f phtsynthesis in WARBURG buffer and in tap water have been made, with the additin f 1.84 X 1 5 M CaD-2. The inhibitin in the WARBURG buffer was fund t be smaller than that in the phsphate buffer (table X1I1).These resultsshw that the bserved discrepancy between the inhibitin f grwth and that f phtsynthesis is at least in part due t a decrease in inhibitin f the latter at high ph values. In Chlrella, n the cntrary, n differences in inhibitin were bserved at different ph values. TABLE XIIT. The inhibitin f phtsynthesis in Scenedesmus at 25 C, upn additin f 1.84 x 1~ 5 M CaD 2 in a carbnate bicarbnate buffer, and in tap water. Carb.bicarb, buffer Tap water Time after additin Rate f phtsynthesis (mm 3 /hr.) N GaD 2 CaD 2 Per cent inhibitin by CaD 2 Rate f phtsynthesis (mm 3 /hr.) N CaD 2 CaD 2 Per cent inhibitin by CaD 2 1 hur 2 hurs 3 hurs The smaller inhibitin f phtsynthesis in the WARBURG buffer may be due t a mre difficult uptake f CaD 2 int the cell frm this buffer. Besides, it is knwn that Scenedesmus is mre tlerant twards a high ph than Chlrella. This fact might explain the slight, but cntinuus decrease in the rate f phtsynthesis bserved in Chlrella, cnfirming PRATT'S (51) results. Such a decrease is nt bserved with Scenedesmus, cnfirming OSTERLIND (44), wh reprted utilisatin f HCOJ as substrate in Scenedesmus quadricauda, whereas this did nt ccur in Chlrella pyrenidsa. 4. INHIBITION IN Chrmatium Earlier experiments with purple sulphur bacteria have been carried ut by GAFFRON (22), VAN NIEL (42), WASSINK et al. (76) and thers. Our experiments were made with a pure culture f Chrmatium indicated as strain D" by ROELOFSEN (54). The rganism was maintained in a malate thisulphate medium in 1 cc glass-stppered bttles with a small jar ver the stpper f the bttle in rder t prevent cntaminatin (EYMERS and WASSINK [14]). As this species is anaerbic, all media had t be biled and cled befre use. The culture medium was based n that f ROELOFSEN (54), as mdified by EYMERS and WASSINK (14) cntaining: 2% NaCl,.1 % (NH 4 ) 2 S 4,.5% K 2 HP 4,.2 % MgS 4 in tap water. It prved t be desirable t use the 2% NaCl partly in pure, partly in crude frm; 1 % f each was used. The ph was brught t 7.4 by additin f 1 N NaOH r 1 N HC1, befre filtratin and sterilizatin. Hereafter,.25cc f a 1% Na 2 S slutin, l.occ f a 1% Na 2 C 3 slutin, ne drp f a 1% H 3 P 4 slutin, 3.75 cc f a 1% sdium malate slutin, and 2.5 cc f a 1% thisulphate slutin were added t cc f the slutin. The species was repeatedly transferred t fresh cultures in shrt intervals, in rder t activate the cells. Hereafter,.5 mm 3 f cells was pipetted int each f the glass-stppered bttles. The rate f phtreductin was measured in the WARBURG apparatus at 29 C in a 1/15 M phsphate buffer with.6% NaCl (ph 7.6). Befre use, ne

53 57(7) 47 day ld cultures were centrifuged, resuspended in the same medium, withut the additin f sdium malate, and shaken in the dark fr a perid f abut 15 hurs in rder t useall the malate left in the cells. Thevesselswere attached t the manmeters, ne f the side tubes was prvided with an utlet during the filling f the vessels (± 3/4 hur). The gas mixture (N2, 3% H and 5% CO2) was passed thrugh an electrically heated reductin ven in rder t remve all xygen left in the mixture. The rate f phtreductin was cmputed by Methd III. Accrding t the frmula CO2 + 2H 2 ->CH 2 + H 2, an uptake rati H2/CO2 = 2 was used, which has been experimentally verified by WASSINK (74). Grwth experiments were made with CaD2 at different cncentratins (,2.19,4.38,6.55,8.73 X 1 6 M, 1.9,1.31, 1.96,2.73 X 1 5 M) f CaD 2. The bttles were placed inthe light cabinet at a light intensity f abut 5 lux at 29 C. Grwth was measured after 1 r 2 days by centrifuging 5 cc suspensin in TROMMSDORFF tubes.the actual activity f the bacteriaafter this treatment was determined by measuring the ratef phtreductin in the WARBURG apparatus. In this case, measurements were made directly, als in the blanks, withut using the prcedure f malate starvatin. The results are summarized in table XIV as an average f 4 replicates. TABLE XIV. The effect f CaD 2 n the rate f grwth and the rate f phtreductin in Chrmatium, as an average f 4 experiments. Cncentratin fcai) a (1- M) Quantity f cells in mm 3 /1 cc after 2 days 1 ) Numerical value') Rel. grwth rate *) Per cent inhibitin Rate f phtreductin mm 3 /hr. Per cent inhibitin *) Initial density.5 mm 3 cells in 1 cc. 2 ) Relative grwth rate = lg. increase f cell density per day It is clear, that the inhibitin f grwth, expressed as percentage f the cntrl, increases with increasing CaD2 cncentratins up t 88 % inhibitin at 2.73 X 1 _5 M CaD2. The inhibitin f grwth in Chrmatium is smaller than that bserved in ther species, the sensitivity f which species is similar t that f Chlrella in hetertrphic grwth. A cmparisn f the results f clumns 4 and 6 in table XIV indicates that phtreductin is still less sensitive and is hardly inhibited at cncentratins at which the grwth rateis strngly decreased. These measurements were made by suspending the cells grwn at the varius CaD-2 cncentratins in fresh phsphate buffer withut CaD2. These findingswerecnfirmed by measuring the inhibitin f phtreductin in shrt timeexperiments upn additin f CaD2f Chrmatiumcells cultivated withut CaD2. The rate f phtreductin was measured during half an hur, after which different cncentratins f CaD? were tipped int the suspensin while the measurement was cntinued until 2-3 hurs after additin (table XV).

54 48 57(7) TABLE XV. The inhibitin f phtreductin f Chrmatium upn additin f CaD 2 in phsphate buffer (ph 7.6), at 5-6 x 1* ergs/cm 2 sec, at 29 C C, as an average f 3 experiments.. Cncentratin in 1~ 5 M CaD 2 mm 3 /hr. Rate f phtreductin Per cent inhibitin It is clear that the range frm the threshld f inhibitin t cmplete inhibitin extends frm 4.4 X 1 5 t 1.29 X 1 4 M CaD 2. Cntrary t the results with Chlrella, inhibitin in Chrmatium was fund t be cnstant within 2 minutes, and it remained cnstant fr at least 3 hurs. The cncentratin at which inhibitin f phtreductin ccurs is much higher than that fr inhibitin f phtsynthesis in Chlrella. 5. EXPERIMENTS WITH Rhdspirillum rubrum, Oscillatria species, Nstc species, Chlamydmnas Rhd spirillum rubrum Grwth experiments with this species were perfrmed in a NaCl-peptne medium, cntaining 1 % peptne,.5 % NaCl, the latter partly in pure, partly in crude frm,.25 % f each being used. Grwth was measured by centrifuging 5 cc suspensin in TROMMSDORFF tubes. N grwth at all was bserved at CaD-2 cncentratins abve 4.38 X 1 6 M CaD2. At lwer cncentratins grwth increases with decreasing CaD2 cncentratins and threshld was fund smewhere between.55 X 1 6 and 1.9 X 1 6 M CaD2. Sme bservatins with a micrscpe revealed that decrease in grwth was crrelated with decrease in mtility f the cells. N mtility at all wasbserved in cncentratins higher than 4.38 X 1 6 M CaD2. Oscillatria species Grwth experiments with Oscillatria were perfrmed in a nutrient slutin cntaining.1% Ca(N 3 )2,.2% K 2 HP 4,.2% MgS 4, 6 X 1 4 % FeS 4. The inculum was intrduced by means f a platinum wire and usually sank t the bttm f the erlenmeyerflask. Grwth ccurred as lng filaments at the surface f the culture medium, rapidly cvering the whle area. It may be mentined here that sme calcium cntaining precipitate always ccurred in the erlenmeyer flasks, in the cntrl aswell as in the flasks with different CaD 2 cncentratins. Only after this precipitate has been disslved by the grwing culture, a vigrus grwth at the surface was bserved. Grwth was estimated in fur degrees, viz, the same rate as the cntrl, mderate grwth, slw grwth and ttal inhibitin f grwth. Experiments were perfrmed with 6 cncentratins f CaD2, viz.,.55, 1.9, 2.19, 3.28,

55 57 (7) X 1~ 6 M CaD2. Frm these estimatins the threshld f inhibitin, 5% inhibitin, and cmplete inhibitin culd be determined with a reasnable accuracy. Inhibitin began t be nticeable at 1.9 X 1 6 M and was cmplete at 3.28 X 1* 6 M CaD2. Oscillatria was reddish brwn t red with defrmed cells in 3.28 and 4.38 X 1 6 M CaD 2. Nstc species The same nutrient slutin was used as fr Oscillatria species, except that.5% Ca(N3)2 was applied instead f.1 % Ca(N3)2- Inhibitin f grwth again ccurred between 1.9 and 3.28 X 1 6 M CaD. Chlamydmnas In these experiments a WARBURG medium was used as nutrient slutin, the cmpsitin f which has been described in Chapter II. Inhibitin ccurred als here between 1.9 and 3.28 X 1 6 M CaD DISCUSSION A survey f inhibitin f grwth, respiratin and phtsynthesis by CaD> in varius plant types as cmpared with Chlrellais represented in table XVI. It is clear that the inhibitin f phtsynthesis and that f grwth shw similar features. Hwever, usually a smewhat strnger inhibitin f grwth was bserved, which may be due t the lng time f expsure t CaD2 (4-8 days). Phtsynthesis was usually measured in shrt time experiments, within ne day (1-3 hurs after additin). The inhibitin range extends rughly between 1" 6 and 1 5 M(~ 1-1 mg. CaD2/l.) fr all the species. Lemna minr, Chrmatium and Chlrella in hetertrphic grwth, are smewhat less sensitive. Phtreductin is far less inhibited than phtsynthesis, while n inhibitin at all was bserved fr endgenus respiratin. The latter was stimulated up t 6 % even at 1 3 M CaD>. Grwth experiments in sand-water cultures with maize and tmates by SCHRIJFSMA (56) and with barley by VERHAGEN (7) revealed that the threshld f inhibitin and especially cmplete inhibitin ccur at higher cncentratins than in Chlrella. Accrding t TAMMES (67) and WEZENBERG (81) CaD> adheres strngly t sil particles. The latter reprted increasing adsrptin in the rder: clay sil, sandy sil and peat mss, which increase was almst prprtinal t the percentage f rganic material f the sil. These high adsrptin values and the fact that inhibitin in higher plants is mre r less restricted t the rt zne prbably explains the smaller inhibitin f grwth in higher plants. A few remarks may be made n the inhibitin f ther aspects f the metablism f Lemna minr. Experiments in ptassium deficient cultures (5) revealed that ptassium deficiency yields an increase in dry weight/frnd. A decrease in dry weight/frnd, hwever, is bserved upn additin f CaD>. The inhibitin by CaD2 therefre is neither due t ptassium remval nr actsin thesameway as ptassium deficiency. Als the lw CaD2 cncentratin at which inhibitin already ccursdes nt supprt theidea f ptassium remval. Anther argument in this directin is given by thefact, that the threshld f KD slubilityis rughly at 1 3 M, which is far higher than the cncentratins at which inhibitin ccurs.

56 5 57 (7) C * " 75 C c c u C ^O.3. 2 IE ^c a E U 5 l lo t 1 1 c X X X fo Tf rs <N TJ : Cs IE c. GJ v T3 > G> S "5.JD 7 E 6 c _ <-> C *-> S O > Q l A * # U ) t «C O O ! 1 X X X X X X X X X Ov^tOcr^Tfccrn an(n-«i- «^* ^- n N 1 D. a > Q CJ c. 2 >>. "5 C _3 "J3 r3 75 c #.3 IE c O «% / N 2 X 75 O x H l» ( < OOC»-^ ^»H ^ X X X *r* Tt r» t* <e ~» r X X X ino\c (N C3 O "2 C3 <N ^" * 5 ^ *nrf X c < > 4 i 4 i 4 < < < t S C -» 1 I > I OOOOOOOOO»-< t < ~m * ^^ ^^ ^««~«"» X X X X X X X X X rr ONr--(N cv I > ' - ^ > i» 1 ^ - ^ - i X X X X X X X X X OOOrl"*'-(S-'n x; x M -E 75 Xj C3 X D. 75 «> r- 5 Q ^O?5 > #c a *»3 X> 73 *-* O <*- c.2 < > C.2 «_ 5 2 c a E c CtS c.2 mm +-* IE.5 x> c-a *-> <*- 75 x: >.E 3 "> CO '5 > # E 75 "c «t 5 l_ O c mm) n ' 5 3 t ^» ^> ^ *- ^ Jt "- «*> *-»; <J<J<J..5. ~ mm (N C c >> 75 O X *-* a 2 ' "S ^ r- 5.1 c ^ 1.3 > = EM ".2 «J» 75 CC c = 75 3 " ) O t? C J O a - c ic i * :» ) ) >! i '-2-2 "^ ^» «s! 5 2 K, >5. s:. ** -S > -^». b. -2 * 5 ' T3 '. C. «. -Si *. 1 & '.-. 3* > =5X5 S 7C. b.c 5 MO««^-«:^ C x;g-c3c ^,.. Q. g «-< r j ^ r O *- X! ^> X! ~ u > O > *--«- >** ' ' X < 1 CL < w ) c > 75 c.2 '>< O u. a e O Urn c Q. u c «>> T3 == fm mm mmm +mt bz a E P"^ mm ^ 52

57 57 (7) 51 The mst striking feature in Lemna minr is the strng inhibitin f grwth and rt frmatin (fig. 21). In cnnectin herewith it may be mentined that VERHAGEN (7) als bserved the first symptms f inhibitin in the rt system, and in barley reprted a lack f frmatin f new side rts, a stunted appearance and a clavate swelling. SCHRIJFSMA (57) bserved a swelling als, and a ramificatin f small maize rts, remaining shrt. She bserved an abnrmal anatmy in rt slides and a yellw discluring f cell wall and prtplast with CaD2. The discluring cmes up t r just beynd the enddermis, therefre theenclsed central cylinder seems t be prtected against penetratin f CaD. CHAPTER VI DISCUSSION AND SUMMARY 1. GENERAL DISCUSSION This investigatin is cncerned with the effects f calcium dipicrylamine (CaD2) n metablic prcesses in varius plant types as cmpared with the actin f ther inhibitrs. The threshld f the txic actin n grwth and phtsynthesis was rughly 1 6 M while cmplete inhibitin was reached at 1 5 M CaD2. This shws clearly that CaD2 is a very pwerful inhibitr, as culd be expected, since armatic nitr-cmpunds, including D, have been used as insecticides fr a lng time. Lwer temperatures still increase cnsiderably the inhibitry effect n phtsynthesis in Chlrella, s that a certain inhibitin was prduced at 12.5 C by rughly ten times lwer cncentratins than thse required at 25 C (figure 7a). The better knwn inhibitrs f phtsynthesis, bth enzyme pisns and narctics, are far less inhibitry (figures 7b,7c, 7d,7e, 7f). With 2,4-dinitrphenl WINTERMANS (83) bserved an increase in inhibitin f phtsynthesis at lwer ph values f the medium (cming finally int the same range as CaD2). A similar effect was bserved with sdium azide, which has been ascribed t the increase in the cncentratin f undissciated mlecules (SIMON and BEEVERS [61, 62], WINTERMANS [83]). Our experiments were made in a carbnate bicarbnate buffer (ph 9.2). Under these cnditins the values fr 5 % inhibitin (table VII) which are the mst accurately determined nes, are in agreement with thse btained by ther authrs (68, 71, 83). The inhibitry effects f hydrgen sulfide (NEGELEIN [41]) and f cpper sulphate (GREENFIELD [27]) appear t be in the same range as that f CaD2. We have, s far, nt studied these inhibitrs. Aside f Chlrella (Chapter III and IV), eight ther plant species frm varius classes have been tested fr their behaviur with regard t CaD2 (Chapter V). The range f inhibitin f grwth in these species and that f auttrphic grwth and phtsynthesis in Chlrella are rughly the same (1' 6 t 1 5 M). Hetertrphic grwth f Chlrella, grwth f Chrmatium and f Lemna minr shw a smaller sensitivity. Mrever, glucse respiratin in Chlrella (figure 15), phtreductin in Chrmatium (table XV) and phtsynthesis in Lemna minr (figure 22) are less inhibited. These facts indicate

58 52 57 (7) that grwth generally is smewhat less sensitive than phtsynthesis. It may be assumed therefre, that grwth f all plant types is inhibited in mre r less the same range, thugh exceptins may be pssible under mre unfavurable cnditins shifting the inhibitin t either higher r lwer cncentratins. In cnnectin with the preceding cnsideratins, the bservatin f WEZENBERG (81) with sugarbeets in water-cultures, f SCHRIJFSMA (56, 57) with maize and tmates and f VERHAGEN (7) with barley in sand-water cultures, may be mentined. They all indicate mre r less the same range f inhibiting cncentratins, thugh cmplete inhibitin ccurred nly at higher cncentratins. In these experiments a strng distrtin f the rts was bserved (58, 7), which is in agreement with ur results with Lemna minr,in which the length and the number f the rts were cnsiderably mre reduced than grwth in general. It is f sme interest t mentin here a few field studies regarding effects f CaD2 n crp plants (6, 64, 81). An imprtant aspectin this case is the high adsrptin f CaD2 in the sil (TAMMES [67], WEZENBERG [81]) which largely depends n the percentage f humus actually present. This fact makes a direct cmparability with the results btained in water-cultures rather difficult. A rugh calculatin may indicate, that the effects bserved in crp yields in the field wuld ccur t much the same extent in water-cultures. Accrding t TAMMES (66), the ttal amunt f pisn required t kill a fish is cnsiderable, thugh fishes die in waste water cntaminated with D already in 1-1 times lwer cncentratins than Chlrella. TAMMES (66) explained this extreme sensitivity by a strng absrptin f D even frm very lw cncentratins, which absrptin accrding t this authr takes place mainly thrugh the gills. Mulds, n the ther hand, are far less sensitive, and n fungistatic effects were bserved at CaD2 cncentratins f the rder f 1 4 M in 14species (SCHOL-SCHWARZ [55]), except in ne species which culd stand nly 1 6 M CaD2. In 8 species even 6 hurs expsures at 1 2 M were survived. The inhibitin with 2,4-dinitrphenl, as already mentined, depends largely n the ph f the medium. Thugh the same effect was expected with CaD2, n significant differences in inhibitin f phtsynthesis n Chlrella were bserved at different ph in a range frm ph 1.4 t 5.9. Hwever, the pk value fr CaD2 is 2.85, whereas that f 2,4-dinitrphenl is 4. (WINTER- MANS [83]). A decrease in extinctin f a slutin cntaining 5.8 X 1 6 M CaD2, which gives abut 75 % inhibitin f phtsynthesis ccurs nly belw ph 4.. Anther difference with 2,4-dinitrphenl is, that in ur experiments with CaD2 n deviatins were bserved in the inhibitin, using 2 and 5cc suspensin liquid, cntrary t the results by WINTERMANS in which in 6 cc a strnger inhibitin than in 3 cc was bserved. It is prbable, therefre, that a still strnger inhibitin f phtsynthesis with CaD2 will nly ccur belw ph 4.. It is nt clear at the mment, why a marked difference in inhibitin was bserved in Scenedesmus in WARBURG buffer as cmpared with tap water. In Chlrella a slight but cntinuus decrease f phtsynthesis was bserved in the cntrl in WARBURG buffer, cnfirming PRATT'S results, whereas n such decrease ccurred in Scenedesmus under the same cnditins, cnfirming OSTERLIND (44). The latter reprted utilisatin f HCO* 3 in Scenedesmus phtsynthesis, whereas this did nt ccur in Chlrella. It seems likely, therefre, that Scenedesmus is mre adapted t high ph than Chlrella. The inhibitin f phtsynthesis with CaD2 is the same in the light limited

59 57 (7) 53 and in the light saturated regin, the pint f transitin remaining the same asin the nn-inhibited suspensins and ccurring rughly at 3.8 X 1 4 ergs/cm 2 sec. and 2.5 X 1 4 ergs/cm 2 sec, at 25 C and 17.5 C respectively. This fact might pint t a narctic actin f CaD>2 n phtsynthesis. Narctic pisning is generally less specific than enzymatic pisning bth with regard t the mlecular structure f the pisn and the cnstitutin f the catalytic systems affected by it. Hwever, the very lw cncentratin f CaD2 (1 6 t 1 _5 M) in which inhibitin ccurs pints t specific interactins with definite catalists rather than t an indiscriminate surface blcking mechanism. Phtreductin in Chrmatium, mrever, is far less inhibited than phtsynthesis. Anther feature against the assumptin f narctic type f pisning is the irreversible inhibitin f phtsynthesis by CaD2. Hwever, it is pssible that in these experiments the expsure t CaDz was t lng r at t high ph values,which may well lead tirreversible injury. Besides thiscad2 can hardly be remved (efficiently nly with methyl-isbutyl ketne). True narctizatin is characterized by reversibility. All tgether, the abve cnsideratins may well rule ut narctic pisning. A decrease in flurescence was bserved with CaD2 in the light saturated as well as in the light limiting range, while n decrease f the CaD2 inhibitin was bserved with an increase f the dark perid in flashing light.these preliminary data suggest that the inhibitin takes place primarily in the phtchemical part f phtsynthesis. The inhibitin f phtsynthesis at light saturatin, hwever, implies that a dark system invlved in phtsynthesis is als affected in rughly the same percentage as that in the phtchemical part. It has been mentined already (Chapter III), that accrding t WELLER and FRANCK, the inhibitin f phtsynthesis with hydrxyl amine is the same at different light intensities, while GAFFRON'S experiments with hydrgen adapted algae made it clear that hydrxyl amine is an enzyme pisn rather than a narctic. The inhibitin with CaD 2 shwssimilar features, and it may be suggested that CaD2 acts in the same way as hydrxyl amine. This view might even seem supprted by LIPSCHITZ' suggestin (37) that armatic nitr-cmpunds in general are reduced via the hydrxyl aminelevel tless pisnus substances. The reductin itself takes place thrugh a sequence f steps. The decrease in inhibitin f phtsynthesis with increase in temperature might pint t an increased reductin f CaD2 t nn-pisnus substances. With hydrxyl amine itself, hwever, a similar temperature effect was nt fund (table V). Regarding the differences in results, btained with hydrxyl amine in this study, and thse described by TAMIYA (68), and WELLER and FRANCK (8), see discussin Ch. Ill, 6, p. 29. We have bserved that a cnsiderable time elapsed befre inhibitin f phtsynthesis by CaD2 reaches a cnstant level (fig. 8). After washing n decrease in inhibitin ccurred. Frm these experiments we cncluded t a cntinuus uptake f CaD2, while, mrever, the inhibitin was irreversible. On the cntrary the inhibitin f the glucse-stimulated respiratin was cnstant within 2 minutes, and remained s hereafter, while the inhibitin was at least partly reversible by washing. As CaD2 is strngly absrbed (67, 81), washing may nt remve the inhibitr cmpletely, Especially at the high cncentratins which are required fr inhibitin f the glucse-stimulated respiratin. It may be assumed, therefre, that the respiratin inhibitin, in principle is reversible. The bserved time f 2 minutes, mrever, was nearly

60 54 57 (7) the same as that fr ther inhibitrs (ethyl urethane, phenyl urethane, hydrxyl amine) with regard t phtsynthesis. Frm experiments n administratin f CaD2 in different flasks at the same time, but starting illuminatin at different mments thereafter (fig. 1), it was cncluded that the increase in inhibitin f phtsynthesis by a cntinued uptake fcad2 ccurs in light nly. This may indicate the inhibitin f an enzyme which is activated by light nly. The increase in inhibitin at lwer temperatures may be ascribed t a decrease in the dark deactivatin. Befre mre can be said definitely abut this suggestin, exact measurements n CaD2 uptake under different experimental cnditins have t be made. Since inhibitin appears already at extremely lw cncentratins, and the determinatin f CaD2 by extractin with methyl-isbutyl ketne was available nly later n in the curse f this investigatin, this fact has nt yet been studied in greater detail. The different aspects bserved with CaD2 cmpared with ther inhibitrs n phtsynthesis and respiratin, may induce us t a further investigatin with this inhibitr. 2. SUMMARY Experiments are described in which the inhibitry effects f calcium dipicrylamine (CaD2) and ther inhibitrs have been investigated in Chlrella and several ther plant species belnging t different classes. The rates f phtsynthesis, phtreductin and respiratin havebeen measured with a WARBURG apparatus, and grwth has been determined by means f centrifuging cell suspensins in TROMMSDORFF tubes, cunting frnds in the case f Lemna, rby visual estimatin in Oscillatria and sme ther algae. CaD2 prved t be a much mre effective inhibitr f phtsynthesis in Chlrella, than ethyl urethane, phenyl urethane, hydrxyl amine, sdium azide, r 2,4-dinitrphenl. The range f inhibitin frm threshld t cmplete inhibitin is rughly between 1 6 and 1 5 M CaD2 and, therefre, is mre cmparable t that reprted fr hydrgen sulphide and cpper sulphate in literature. Inhibitin f auttrphic grwth in Chlrella was cmparable t that f phtsynthesis, while hetertrphic grwth in Chlrella, and grwth, and especially phtreductin in Chrmatium are less inhibited. This prbably means that auttrphic grwth in Chlrella is limited by a shrtage f assimilates. Cntrary t the high sensitivity f grwth and phtsynthesis, n inhibitin, but a stimulatin up t 6% was fund even at 1 _2 M CaD2 in respiratin. Glucse-stimulated respiratin was 5% inhibited at this cncentratin. This inhibitin was at least partly reversible. The same inhibitin percentage was bserved in Chlrella phtsynthesis under cnditins f light saturatin as well as under thse f light limitatin, the inhibitin was fund t be nn-reversible by washing. Phtreductin in Chrmatium was cnsiderably less sensitive than Chlrella phtsynthesis. These facts d nt supprt a purely narctic actin f this pisn, but rather pint t a specific type f inhibitin. Sme flashlight experiments revealed that inhibitin was scarcely influenced by lnger dark perids. Flurescence measurements clearly shwed the change in yield at lw as well as at high light intensities. The generally high acceptance f energy by armatic nitrcmpunds prbably explains the decrease bserved in yield f flurescence upn additin f CaD2.

61 57 (7) 55 A strng increase in the inhibitin f phtsynthesis was bserved with CaD2 at lwer temperatures, which did neither ccur with ethyl- and phenyl urethane nr with hydrxyl amine. Fr an explanatin ne culd think f a decrease in the dark deactivatin f the CaD 2 sensitive enzyme. Thugh the time required t btain equilibrium was cnsiderable, especially in carbnate bicarbnate buffer (1-2 hurs), n definite differences in time required fr reaching the equilibrium were bserved at varius temperatures. The inhibitin f grwth f Lemna arrhiza, Scenedesmus, Nstc spec, Rhdspirillum rubrum, Oscillatria spec, and Chlamydmnas by CaD2 ccurred rughly in the same range f cncentratins as that f auttrphic grwth in Chlrella. Chrmatium and Lemna minr are smewhat lesssensitive. The rts f the latter, hwever, are a very sensitive indicatr fcad«2. Thugh CaD2 has a marked influence n grwth and phtsynthesis f species in water cultures, CaD2 is much less harmful under field cnditins wing t the high adsrptin capacity f the sil. This has been cnfirmed in field experiments. Only at very high artificial increase f the cntaminatin a decrease in yield was bserved. 3.SAMENVATTING De werking van calcium dipicrylamine p planten is van belang in verband met de mgelijkheid van tepassing van deze stf bij bereiding van kaliumhudende meststffen. De remming van verschillende stfwisselingsprcessen dr calcium dipicrylamine (CaD2) en andere remstffen werd nagegaan bij Chlrella, Lemna minr, Lemna gibba, Lemna arrhiza, Chrmatium, Scenedesmus, Nstc spec, Rhdspirillum rubrum, Oscillatria spec, en Chlamydmnas. De phtsynthese snelheid, de phtreductie en de ademhaling werden gemeten in een WARBURG apparaat, de greisnelheid dr centrifugeren van eel suspensies in TROMMSDORFF buisjes, dr telling van het aantal schijven {Lemna),f dr een schatting van de tename {Oscillatria, Nstc). CaD2 remt de phtsynthese reeds in zeer lage cncentraties ( M, fig. 7a). Ethyl urethaan (fig. 7b), phenyl urethaan (fig. 7c), hydrxyl amine (fig. 7d), 2,4-dinitrphenl (fig. 7e) en natrium azide (fig. 7f) daarentegen zijn pas in hgere cncentraties werkzaam. De auttrphe grei van Chlrella, Lemna arrhiza, Scenedesmus, Nstc spec, Rhdspirillum rubr., Oscillatria en Chlamydmnas wrdt min f meer bij dezelfde Q1D2 cncentraties geremd als de phtsynthese, tabel XVI geeft hiervan een verzicht. Overeenkmstige resultaten werden verkregen met bieten in water-cultures (81), mais, tmaten engerst (57,7)in zand-cultures. Vral de wrtelgrei was sterk geremd (58,7). In Lemna minr werd de wrtelgrei vlledig geremd bij CaD2 cncentraties, waarin andere prcessen ng nageneg geen vergiftigingssymptmen te zien gaven. De hetertrphe grei van Chlrella en Chrmatium, maar vral de phtreductie van Chrmatium zijn minder gevelig. In tegenstelling hiermee werd een stimulatie van de ademhaling waargenmen, welke zelfs bij 1 2 M CaD2 ng 6% bedreg. Bij de glucse-ademhaling werd bij deze cncentratie een remming van 5 % waargenmen, welke in tegenstelling tt die van de phtsynthese gedeeltelijk reversibel is na uitwassen. Over het algemeen werd, met uitzndering van CaD2, reeds 2 minuten na teveging van de remstf een cnstante remming van de

62 56 57 (7) phtsynthese waargenmen. Afhankelijk van de cncentratie nam de phtsynthese remming met CaD2 in de tijd te. Deze tename werd verklaard dr een cntinue pname van CaD2, welkealleen in het licht plaatst vindt (fig. 1). De resultaten van de glucse ademhaling, waarin k na ngeveer 2 minuten een cnstante waarde van de remming werd bereikt, zijn hiermee in vereenstemming. Zwelbij lichtlimitering als bij lichtverzadiging van de phtsynthesewerden vereenkmstige remmingspercentages waargenmen. Hewei deze waarneming een aanwijzing is vr een narctische werkingvan deze remstf, is aan te nemen, dat CaE>2een specifiek enzym vergif is, dat behalve bepaalde dnkerreacties k de energieverdracht bij de phtsynthese aangrijpt, de vrlpigechlrphylflurescentie en flitslicht preven wijzen k in deze richting. Bij de laatste werd geen vermindering van de remming waargenmen bij een langere dnkerperide, terwijl de flurescentie zwel bij lichtverzadiging als bij lichtlimitering verlaagd werd. Bij lagere temperaturen werd een sterke tename van de phtsynthese remming met CaD2 in Chlrella waargenmen. Dit effect van de temperatuur werd niet bij phenyl- en ethyl urethaan en bij hydrxyl amine gecnstateerd en is vralsng niet geheel te verklaren. Hewei, zals reeds eerder vermeld, de grei en de phtsynthese reeds bij zeer lage cncentraties geremd wrden is een pbrengstvermindering in het veld bij kalibemesting met deze stf als verntreiniging vrshands niet te verwachten dr enerzijds de hge adsrptie van CaD2 in de grnd,speciaal aan humus, en anderzijds de geringe verntreiniging van het gehele veld. ACKNOWLEDGEMENT This wrk was carried ut in thescpe f a research grup subventedby the N.V. Mekg, IJmuiden at the Labratry f Plant Physilgical Research f the Agricultural University, Wageningen. The authr is indebted t the N.V. Mekg fr the pssibility t publish the results btained with calcium dipicrylamine and t Prfessr E. C. WASSINK fr his advice and criticism. REFERENCES 1. ARNON, D. J., Micrelements in culture-slutin experiments with higher plants. Am. J. Bt. 25, (1938). 2. ASHBY, E. T. and A. OXLEY, The interactin f factrs in the grwth f Lemna. VI. An analysis f the influence f light intensity and temperature n the assimilatin rate and the rate f frnd multiplicatin. Ann. Bt. 49, (1935). 3. AUDUS, L. J., Studies n the ph relatinships f rt grwth and its inhibitin by 2,4- dichlrphenxy acetic acid and cumarin. New Phytl. 48, (1949). 4. BEEVERS, H., 2,4-Dinitrphenl and Plant respiratin. Am. J. Bt. 4, (1953). 5. BIERHUIZEN, J. F., Observatins n ptassium deficiency in Lemna minr L. Med. Landb. Hgesch. Wageningen/Ned. 54 (7), (1954). 6. BIERHUIZEN, J. F. and H. J. WEZENBERG, Reprt t Mekg, IJmuiden. 7. BLACKMAN, G. E., Studies in the principlesf phyttxicity. I. The assessment f relative txicity. J. Expt. Bt. 3, 1-27 (1951). 8. BRADBURRY, D. and W. B. ENNIS, Jr., Stmatal clsure in kidney bean plants treated with ammnium 2,4-dichlrphenxyacetate. Am. J. Bt. 39, (1952). 9. BROWN, A. W. A., Insect cntrl by chemicals. Edited Jhn Wiley and Sn, New Yrk., 817 p. (1951).

63 57 (7) BROWN, H. T. and F. ESCOMBE, Researches n sme f the physilgical prcesses f green leaves w'th special reference t the interchange f energy between the leaf and its surrundings. Prc. Ry. Sc. Lndn series B, 76, (195). 11. BROWN, J. W., Effect f 2,4-dichlrphenxyacetic acid n the water relatins, the accumulatin and distributin f slid matter, and the respiratin f bean plants. Bt. Gaz. 17, (1946). 12. BROWN, J. W. and R. L. WEINTRAUB, A leaf repressin methd fr evaluatin f frmative activity f plant grwth regulating chemicals. Bt. Gaz. Ill, (195). 13. EMERSON, R. and W. ARNOLD, A separatin f the reactins in phtsynthesis by means f intermittent light. J. Gen. Phys. 15, (1932). 14. EYMERS, J. G. and E. C. WASSINK, On the phtchemical carbn dixide assimilatin in purple sulphur bacteria. Enzyml. 2, (1938). 15. FEIGL, F., Qualitative analysis by spt tests. Edited Elsevier Amsterdam, 462 p. (1939). 16. FRANCK, J., The Relatin f the Flurescence f Chlrphyll t Phtsynthesis. In Phtsynthesis in plants (1949). 17. FRANCK, J., C. S. FRENCH and T. T. PUCK, The flurescence f chlrphyll and phtsynthesis. J. Phys. Chem. 45, (1941). 18. FRANCK, J. and K. F. HERZFELD, Cntributin t a thery f phtsynthesis. J. Phys. Chem. 45, (1941). 19. FREELAND, R. O., Effects f 2,4-D. and ther grwth substances n phtsynthesis and respiratin in Anacharis. Bt. Gaz. Ill (3) (195). 2. FREUNDLICH, H., Ober die Adsrptin in Lsungen. Zs. f. physikal. Chem. 57, (197). 21. GAFFRON, H., Ober auffallende Unterschiede in der Physilgie nahe verwandter Algenstamme, nebst Bemerkungen iiber Lichtatmung. Bil. Zentr. 59, (1939). 22. GAFFRON, H., Cber die Khlensaure-assimilatin der rten Schwefelbakterien. Bich. Zs. 269, (1934). 23. GAFFRON, H., The effect f specific pisns upn the phtreductin with hydrgen in green algae. J. Gen. Phys. 26, (1943). 24. GAVAUDAN, P., H. POUSSEL and M. DODE, Cmparaisn du puvir mit-inhibiteur des substances de la serie armatique en functin de Tactivite thermdynamique de leurs slutins. C.R. d.l. Sc. Bil. 138, (1944). 25. GENDEREN, H. VAN, Txicity f crps grwn n KD treated sil. I. Feeding experiments with rabbits and pigs. Scient. Reprt C 89, Mekg, Dmuiden (1952). 26. GORHAM, P. R., Grwth factr studies with Spirdella plyrrhiza (L) Schleid. Am. J. Bt. 32, (1945). 27. GREENFIELD, S. S., Inhibitry effects f inrganic cmpunds n phtsynthesis in Chlrella. Am. J. Bt. 29, (1942). 28. HEUBNER, W., Giftung armatischer Nitrverbindungen. Arch. f. Expt. Path. u. Pharm. 25, (1947). 29. HILLE, J. C. VAN, The quantitative relatin between rate f phtsynthesis and chlrphyll cntent in Chlrella pyrenidsa. Rec. Trav. bt. Need., 35, (1938). 3. HOLZER, H., Phtsynthese und Atmungskettenphsphrylierung. Zs. Naturfrsch. 6b, (1951). 31. JOHNSON, M. J., Manmetric technics and related methds fr the study f tissue metablism by UMBREIT, W. W., R. H. BURRIS and J. F. STAUFFER. Minneaplis Burgess Publ. C KAUTSKY, H. and A. HIRSCH, Chlrphyllflureszenz und Khlensaure-assimilatin. Das Flureszenzverhalten griiner Pflanzen. Bich. Zs. 274, (1934). 33. KAUTSKY, H. and H. SPOHN, 1. Apparatur zur vergleichende Messung der Flureszenzanderungen lebender Blatter. 2. Der Einflusz der Temperatur auf die Flureszenzkurve. Bich. Zs. 274, (1934). 34. KELLY, S. and G. S. AVERY, The effect f 2,4-dichlrphenxyacetic acid and ther physilgically active substances n respiratin. Am. J. Bt. 36, (1949). 35. KOK, B., Sme sensitive and recrding vlumeters. Bich. Biphys. Acta, 16, (1955). 36. LINDEMAN, W., Over de betekenis van phsphaat in de phtsynthese van Lemna minr L. Diss. A'dam, 1-83(1952). 37. LIPSCHITZ, W. L., Giftung und Entgiftung armatischer Nitrverbindungen. Arch. f. Expt. Path. u. Pharm. 25, (1948). 38. NAKAMURA, H., Ober die Khlensaure-assimilatin bei niederen Algen in Anwesenheit des Schwefelwasserstffs. Acta Phytchim. Japan, 1, (1938).

64 58 57 (7) 39. NAKAMURA, H., Ober den Einflusz der Blausaure auf die Phtsynthese vn Scenedesmus. Acta Phytchim. Japan, 1, (1938). 4. NANCE, J. F., Inhibitin f salt accumulatin in excised wheat rts by 2,4-dichlrphenxyacetic acid. Science, 19, (1949). 41. NEGELEIN, E., Ober die Wirkung des SchwefelwasserstfFs auf chemische Vrgange in Zellen. Bich. Zs. 165, (1925). 42. NIEL, C. B. VAN, and E. M. MULLER, On the purple bacteria and their significance fr the study f phtsynthesis. Rec. Trav. Bt. 28, (1931). 43. ORANJE, P. J., Gasentladungslampen. Philips Technische Biblitheek, 294 p. (1943). 44. OSTERLIND, S., Inrganic carbn surces f green algae. III. Measurements f phtsynthesis in Scenedesmus quadricaudaand Chlrella pyrenidsa. Phys. Plantarum 4 (2),* (1951). 45. PAAUW, F. VAN DER, The indirect actin f external factrs n phtsynthesis. Rec. trav. bt. neerl. 29, (1932). 46. PJRSON, A., and F. SEIDEL, Zell und stftwechselphysilgische Untersuchungen an der Wurzel vn Lemna minr L. unter besnderer Berucksichtigung vn Kalium und Kalziummangel. Planta 38 (4), (195). 47. PIRSON, A., C. TICHY and G. WILHELMI, Stffwechsel und Mineral Salzernahrung einzelliger Grunalgen. I. Vergleichende Untersuchungen an Mangelkulturen vn Ankistrdesmus. Planta 4, (1952). 48. POTTER, C. and E. M. GILLHAM, Effects f atmspheric envirnment, befre and after treatment n the txicity t insects f cntact pisns. Ann. Appl. Bil. 33, (1946). 49. PRATT, R., Studies n Chlrella vulgaris. VI. Retardatin f phtsynthesis by a grwth inhibiting substance frm Chlrella vulgaris. Am. J. Bt. 3, (1943). 5. PRATT, R., Studies n Chlrella vulgaris. VII. Influence f the age f the culture n the rates f phtsynthesis and respiratin. Am. J. Bt. 3,44-48 (1943). 51. PRATT, R., Studies n Chlrella vulgaris. VIII. Influence n phtsynthesis f prlnged expsure t sdium bicarbnate and ptassium bicarbnate. Am. J. Bt. 3, (1943). 52. RABINOWITCH, E. I., Phtsynthesis and related prcesses. Vl. I. Interscience Publ. Inc. New Yrk, 559 p RABINOWITCH, E. I., Phtsynthesis and related prcesses. Vl. II. part 1, Interscience Publ. Inc. New Yrk, p , ROELOFSEN, P. A., On phtsynthesis f the Thirhdaceae. Diss. Utrecht, SCHOL-SCHWARZ, M. B., Txicity f dipicrylamine fr sil rganisms. Scient. Reprt C 87, Mekg, IJmuiden (1952). 56. SCHRIJFSMA, A. S. C., Txicity f dipicrylamine fr crps. Reprt 2, Txic cncentratin f CaD 2 and pisning symptms fr maize and tmat. Scient. Reprt C 53, Mekg, IJmuiden (1952). 57. SCHRIJFSMA, A. S. C, Txicity f dipicrylamine fr crps. Reprt 3, Anatmic abnrmalities in pisned maize rts. Scient. Reprt C 54, Mekg, IJmuiden (1952). 58. SEEL, H., Beitrage zur Txlgie einiger Schadlingsbekampfungsmittel aus der Gruppe armatischer Nitrverbindungen. Arch. expt. Path. u. Pharmacl. 27, (1949). 59. SEIFERT, F., Bestimmung vn Trinitrtlul und Hexanitrdiphenylamin neben einander in Wasser und Abwasser. Wasser 17,89 (1949). 6. SHIBATA, K. and E. YAKUSHIJI, Der Reaktins Mechanismus der Phtsynthese. Naturwissensch. 21, (1933). 61. SIMON, E. W. and H. BEEVERS, The effect f ph n the bilgical activities f weak acids and bases. 1. The mst usual relatinship between ph and activity. New Phytl. 51, (1952). 62. SIMON, E. W. and H. BEEVERS, The effect f ph n the bilgical activities f weak acids and bases. 2. Other relatinships between ph and activity. New Phytl. 51, (1952). 63. SMITH, F. G., C. L. HAMNER and R. F. CARLSON, Changes in fd reserves and respiratry capacity f bind weed tissues accmpanying herbicidal actin f 2,4-dichlrphenxyacetic. acid. Plant Physil. 22, (1957). 64. SPIERINGS, F., in preparatin. Reprt t Mekg, IJmuiden. 65. SPRUIT, C. J. P. and E. C. WASSINK, The simultaneus bservatin f xydatin-reductin ptentials and chlrphyll flurescence f Chlrella suspensins. Bich. et Biphys. Acta 15, (1954). 66. TAMMES, P. M. L., Observatins n the effect f temperature n the balance between entry f a pisn and the prcess f detxificatin. Scient. Reprt, Mekg, IJmuiden.