The Relation of the Membrane Potential of Nitella to Photosynthetic Processes and External Conditions

Transcription

1 The College t Brockport: Stte University of New York Digitl Environmentl Science nd Ecology Theses Environmentl Science nd Ecology The Reltion of the Membrne Potentil of Nitell to Photosynthetic Processes nd Externl Conditions Shy-Whey Mrgret Koh The College t Brockport, skoh@umrylnd.edu Follow this nd dditionl works t: Prt of the Botny Commons Repository Cittion Koh, Shy-Whey Mrgret, "The Reltion of the Membrne Potentil of Nitell to Photosynthetic Processes nd Externl Conditions" (1973). Environmentl Science nd Ecology Theses This Thesis is brought to you for free nd open ccess by the Environmentl Science nd Ecology t Digitl It hs been ccepted for inclusion in Environmentl Science nd Ecology Theses by n uthorized dministrtor of Digitl For more informtion, plese contct kmyers@brockport.edu.

2 THE RELATION OF THE MEMBRANE POTENTIAL OF NITELLA TO PHOTOSYNTHETIC PROCESSES AND EXTERNAL CONDITIONS A Thesis Presented to the Grdute School of the Stte University of New York College t Brockport As prtil fulfillment of the requirements for the Degree of Mster of Science By Mrgret Shy-tJhey Koh August J_2}3

3 "No informtion derived from this thesis my be published without permission of the originl uthor, with whom copyright lies."

4 t s me ce I cme 1 ws

5 TABLE OF CONTENTS INTRODUCTION A. B. c. D. Origin of the resting membrne potentil.. The role of the H + extrusion pump Possible mechnisms of H + extrusion.... Fctors ffecting the membrne potentil OBJECTIVES AND EXPERIMENTAL STRATEGIES A. Chnges in externl environment. B. Injection MATERIAL: Nitell Culturing nd Cell Selection.. 19 METHODS A. Membrne Potentil Mesurements... B. Illumintion by Monochromtic Light C. Injection:.of Solutions into Cells. D. C0 2 Fixtion E. T5les 1 nd 2, Solution Compositions Tble 3, Light Mesurements RESULTS AND DISCUSSION A. Chnges in Externl -Environment 1. Increse in internl ph expected Devrese in internl ph expected B. Injection of Mterils into Cells C. Tbles 4 through D. Figures 1 nd CONCLUSIONS SUMMARY.. LITERATURE CITED

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32 TABLE 1 COMPOSITION OF NITELLA CULTURE SOLUTION 3 0 mm CC mm MgCl mm KCl 2.0 mm N.HC mm KN mm NH 2 P mm tris,- (hydroxymethyl) - minoeth.ne neutrlized with HCl to ph ml/liter micronutrient stock solution Micronutrient stock solution: filtrte from 50 g Brockport brown soil boiled 30 min. in 1.0 liter 6.0 mm ethylenedimine tetr.cette t ph 8e0.

33 0.03 TABLE 2. COMPOSITION OF SOLUTIONS. Concentrtions re millimoles per liter. KOH to ph 7.2 nd used immeditely The K ph 7.2 solution ws neutrlized with Ion or K K Low High co 2... co2-2n 2N + Moleculr Crbon Crbon testing testing COz + co 2 ph co HC Cl so H 2 Po , Oo l o , 0.5 N K l o O Mg c "I A i.,u (). lev (\ 1 1:\.,$ Ve.J-.J.J

34 TABLE 3 ENERGY FLUX DENSITY OF MONOCHROMATIC The desired energ9 flux densities t ll the wvelengths give the quntl flux density of 1.5 x 10 - Einsteins/cm2sec (nm) Energy flux density desired $rgs/cm 2 sec) Energy flux density mesured ±400 ±400 ±300 ±300 i- 300 ± ±300 (Ergs/cm 2.. sec) Qu.nt.l flux le density clibrted ±0.15 ±"0.15 :!:"Oe15 ±0@15 ± "± ± :to.1s (Einsteins/cm2sec X 10 9)

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47 TABLE 4. EFFECT OF A ph INCREASE FROM 5.7 TO 7.2 ON MEMBRANE POTENTIAL WITH WITHOUT BICARBONATE PRESENT ph 7.2. For the sequence of solution chnges red from left to right in the tble. The K ph solution ws neutrlized.with KOH to ph 7.2 nd used innneditely in the experiment. - Low Crbon ph 7.2 solution hd 0"2 mm Hco 3 dded to it. See Tble 2 for the complete compositions. Cells with typicl responses re shown on the bottom of the tbleo The "prime" designtion ppended to n Exp,. No.. mens tht it is the second tril on one cell. (Act. Pot. =Action Potentil, Low C = Low Crbon). ' -mv Exp. No. 26d' 2 26e g 3 Cell Age, Dys K ph 5e Mx. K ph Act.. Finl K Mx Act.. Finl Pot.. ph 7.2 Low C Pot. Low C ph ph 7o Finl K ph Finl K ph j m 8 26m' 9 MEAN ± SEM :t" "i" :t-5 195:!:" ± ± i- 7 Cells with Atypicl Responses 26d 1 26h 5 26i 7 26k

48 TABLE 5. EFFECT OF LIGHT INTENSITY ON THE MEMBRANE POTENTIAL (2N, LOW CARBON, HIGH CARBON AND K. S The light intensity ws chnged from 45 ft-c (cool-white fluorescent light) to 200 ft-c (45 ft-c cool-white ft-c incndescent)., The membrne potentils under the higher intensity light stbilized fter bout 50 minutes; these re the potentils listed. The cells in the High Crbon solution t ph 5.7 were conditioned in K ph 5.7 solution; the membrne potentils of the cells in the K solution under 45 ft-c before the strt of the experiment re lso listed.. The other cells were conditioned in the sme solutions s used in the experiments. For the sequence of light conditions used, red from left to right in the tble. (Low C = Low Crbon, High C = High Crbon).. Em, -mv Exp. Exp.. Cell Age, 200 ft-c 45 ft-c So ln.. No. D.ys 3lb f N ph 8 o.3 32b c d b MEAN ± SEM g 136 :±"4 29d f Low C 29h ph 7 o b c MEAN± SEM 198 :±" K ph ft-c High C 44 4 (173) ph b 2 (159) c 5 (170) ± SEM K 45b c MEAN

49 TABLE 6. EFFECT OF 675 run AND 715 nm MONOCHROMATIC LIGHT ON THE MEMBRANE POTENTIAL. (2N ph 8.3 SOLUTION). The superscript pended to the E vlue indictes the sequence of lighting conditions. rw Exp. 31e E vlue in 45 ft-c cool-white ws used s stndrd nd ws m checked before ech lighting condition nd fterwrds. in 45 ft-c listed is the men vlue obtined. In Exp. 42 nd 42b, Em vlue in drk ws checked before nd fter ech monochromtic wvelength, with the Em vlue in 45 ft-c only being mesured t the strt of ech experiment. The Em vlue in drk shown in tble is the men obtinedo In Exp. 31, 42, nd 42b the cells were lso subjected to the C0 2 ddition t 675 nm; 675 nm nd 715 nm; drk, 675 nm nd 715 nm, respectively; those results re shown in Tble 11. The durtion of ech tretment ws bout 90 minutes. The intensity of the monochromtic light ws (1.5 t Ool5) Einsteins/ cm 2 sec, which ws equivlent to 2600 Ergs/cm 2 sec for 675 nm. -mv The Em vlue Exp., Noo Cell Age, Dys 45 ft-c,drk 675 nm 715 nm 31e b

50 TABLE 7. THE DEPOLARIZING EFFECT OF VARIOUS WAVELENGTHS OF MONOCHROMATIC LIGHT AS COMPARED TO DARKNESS (2N ph 8.3 SOLUTION). These results were obtined in two experiments on the sme N ell internodl cells. E m vlues in drkness were mesured both before the monochromtic light sequence nd fterwrds. The drk vlue corresponding to given monochromtic light vlue ws obtined by liner interpoltion of the two drk vlues, i.e. liner chnge in the drk vlue with time ws sumed. The rdint flux density ws (1.5 ± 0.15) x 10- Einsteins/cm 2 sec; this corresponis to 3900 ± 400 ergs/cm2 sec for 440 nm light nd ergs/cm 2 sec for 675 nm. Sequence Errh Depolr- Sequence Em, p pql t'nm -mv iztion, mv -mv iztion, mv DARK before 0 before DARK fter fter 176 0

51 TABLE 8. EFFECT ON THE MEMBRANE POTENTIAL OF ADDITION OF C02 + Hco 3 - TO K ph 5.7 SOLUTION AT SAME ph The High Crbon ph 5e7 solution contins lo mm co 2 nd 0. 2 mm HC0-3 with the Hco3 - replcing Cl - in the K ph 5. 7 so1utiono See Tble 2 for complete compositions. TheA Em ws obtined by subtrcting the men of the two vlues for the K solution from the vlue of the High Crbon solution. The "prime" designtion ppended to Experiment No. mens tht it is the second tril on the sme cell, which ws done fter letting the cell rest overnight in K solution ph 5.7. For the sequence of solution chnges red from left to right. (High C = High Crbon) Em, -mv Expo Cell Age, K Mxo High Finl High Finl, K AEro, No.. Dys c ph 5. 7 c ph ph 5.7 mv ' b c d f g h MEAN t SEM 154 :t ,

52 . TABLE 9.. THE EFFECT OF C0 2 ON THE :MEMBRANE POTENTIAL NEUTRAL ph (C0 2 -TESTING SOLUTION)@ C0 2 ws bubbled into the C0 2 -testing solution t ph contining mm Hco The lowering of the ph to by the C0 2 ddition is in itself ssumed to hve little or no effect on the membrne potentil. See text nd Tble 2 for further detils.a JSn ws obtined by subtrcting the men of the two vlues tor the ph 7.,6 solution from the vlue t ph 6.,8., E m, -mv Exp., Age, Cell Light ph 7 6. ph6.,8 ph 7.,6 b.e,m No., Dys Intensity, (C02) ft-c b d " 29e " H 29g u 29h MEAN± SEM for 200 -c 188±9 186±"22 189±10

53 TABLE 10. THE TRANSIENT HYPERPOLARIZING EFFECT OF co 2 ON THE :MEMBRANE POTENTIAL OF NITELLA CELLS IN LIGHT (2N SOLUTION)o The 2N solution contins 2 rom N, ool mm K nd 1.0 mm HC The co 2 is bubbled into the 2N solution, lowering the ph from 8.3 to 6.5 nd giving H 2 C0 3 concentrtion of 0. 8 mj1. The complete solution compositions re given in Tble 2o E m, -mv Exp. Cell Age, Noo Dys Intensity, ft:c Mx., N + C0 2 Finl 2N + COz 2N ph 8o3 31 '6 3ld died 162 Em, -mv t 45 ft-c 3lb b 10 32c 6 32d H MEAN± SEM t 200.ft-c H n u ± :t " :!

54 TABLE 11. THE EFFECT OF C0 2 ON THE MEMBRANE POTENTIAL OF NITELLA CELLS IN MONOCHROMATIC RED AND FAR RED LIGHT (2N SOLUTION)., 2N solution contins 2 mm N, 0.1 mm K nd 1.0 mm HC03 -. The C02 is bubbled into the 2N solution, lowering the ph from 8o 3 to 6.5 nd giving H 2 COJ concentrtion of 0.8 mm. The rdition flux density is given in Tble 3 nd the solut on compositions re given in Tble 2o The potentils t ph 6.5 were the mximum nd the stedy stte levels obtined, the ltter t bout 2 hours fter the C02 ddition. E m, -mv Expo Cell Age, Wvelength, 2N Mximum Finl ---2N No., Dys nm ph N + COz 2N + C0 2 ph 8.3 _ 6oS ph 6e b b b 6 DARK

55 TABLE 12,. THE EFFECT OF INJECTING VARIOUS SUBSTANCES INTO THE VACUOLE OF NITELLA CELLS: CHANGES IN THE MEMBRANE POTENTIAL. See Methods for detilso The uprime" designtion ppended to n Experiment No.. mens tht it is the second injection for tht cell.. (Inj. = Injection).. ' -mv Expo Cell Substnce Injection Durtion, Approx.. Drk or Before At End Arter No. Age, Injected Rte, hr Finl Cone. Light Inj.. Of Inj.. Inj.. Dys ml/hr in Cell, mm Period 7 2 Distilled II 7b 5 HftO 3.8 4o5 " p L 0.. L Se M 300 Oo o4 D ( DL Glycer- 2o D ' 2 ldehyde o 2 D ( phosphoric + 2o5 1o25 cid ph 2.,0 fl 1l L l' 9 " L H 15b ,0 7o 2 L b' M L c 4 DL Glycer- 2o o4 L ldehyde-3- phosphte, K slt ph 6o mm Di- 2o Oo008 L hydroxy Oo008 L 154 cetone 8b 6 H (this 3o L cell conditioned D-4 solution 16 7 Oo0434 M 3-2o L (2 16b 3 phosphogly- " Oo9 10 L n 16c 9 eerie cid 1 11 L n 16d 6 5" L u 16f L OS mm eve OoQ3 L

56 " 40 +J 30 Ill N ttl...-i 0 p.. Q) 0 <D <2> Wvelength, nm Fig. 1. Depolrizing effect of vrious wvelengths of monochromtic light on Nitell,s compred to drkness,in 2 N ph 8,3 solution. The numbers on the curve indicte the sequence of the monochromtic wvelengths pplied. The uncircled mnubers indicte the vlues obtined from 1.0-dy old cell while the circled ones indicte those obtined from the sme cell on dy 12. Between dys 10 nd 12, the ce 11 ws kept under 45 footcndles cool white fluorescent light. The precise vlues obtined re presented in Tble 7.

57 z!4-3.o Rdint Flux Density, 2 Kergs/ em sec Fig. 2. The dependence of the level of the membrne potentil on the intensity of 675 nrn light. The results were obtined on 10-dy old Nitell cell in 2 N ph 8.3 solution.

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