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Apparatus for kinetic studies of carbon fixation in leaf tissue
.\NALYTIC!AL
46,
BIOCHEMISTRY
Apparatus
for
250-260
(1972)
Kinetic
Studies
in Leaf J. R. BEDBROOK, Department
of Cell
D.
Biology,
VAN
Received
AND
R. E. F. MATTHEWS
of Auckland, July
Fixation
Tissue
SLUIS,
University
of Carbon
Auckland,
New
Zealand
28, 1971
The most detailed studies on the path of carbon fixation in phot.osynthesis have been carried out with algae, where flow methods can conveniently be applied (1). Carbon dioxide fixation in leaves has usually been studied using labeling periods of minutes or hours, and relatively simple devices for exposure of the tissue to CO, (2-7). Hatch and Slack (8) and Hatch et al. (9) used a simple Perspex box which could accommodate six leaf segments. Individual segments could be removed from the box at different times. We recently began to study the effects of virus infection on the path of carbon fixation in leaf tissue. For this work we needed to be able to expose small pieces of healthy and virus-infected leaf to ‘“CO, under identical conditions for a range of short labeling times, We describe here an apparatus which allows eight pieces of leaf tissue to be exposed to ‘“COz in a single working chamber. The ‘“COz is generated in a subsidiary chamber and transferred to a holding chamber by means of a piston. At the beginning of the labeling period the piston is used to force the ‘“CO, rapidly and uniformly through a manifold into the working chamber. The labeling period for each sample can be varied independently down t.o about one second. MATERIALS
AND
METHODS
General. Chinese cabbage (Brassica pekinensis Rupr.) var. Wong Bok and corn (Zea mays L.) were grown in the glasshouse under conditions of natural lighting. A rectangular block of brass was milled to make a cutter 2 cm X 1 cm, which was used to take leaf samples of uniform area. For labeling experiments leaf samples held at a temperature of 25-27” were exposed to light of 2000-2200 lumensjsq ft from a Philips HLRG 400 W horticultural lamp. l”COz was generated from 14C-labeled barium carbonate (CFA 431, Radiochemical Centre, Amersham, England). Description of apparatus. The apparatus is illustrated in Fig. 3 and 250 @ 1972 by
Academic
Press,
Inc.
APPARATCS
FIG.
FOR
1. General
CARRON
view
251
FIXATION
of the
assembly.
nL .=
L1 FIG. 2. Front see text.
elevation
of
apparatus
showing
E
completed
assembly.
For
details
252
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
FIG. 3. Working drawings showing drilling of Perspex block. Two vertical sections of the block are shown. The upper vertical section is drawn through the center of the working chamber. The lower vertical section is drawn through the center of the ‘“CO, generating chamber and shows the piston and manifold for distributing the gas to the working chamber.
shown in front elevation in Fig. 2. A solid block of Perspex (poly(methy1 methacrylate), Imperial Chemical Industries Ltd.) is machined and bored to form a working chamber (A), which is closed at both ends with a stainless-steel cap (B). The caps are provided with gate taps. The working chamber is pierced vertically with eight channels which in turn contain eight tissue holders (numbered l-8 in Fig. 2). These are sealed with a cap (C) and an O-ring. The O-ring also acts as a friction brake. The piece of tissue is held in place by three metal hooks (shown
APPARATUS
FOR
CARBON
FIXATION
253
in Fig. 4C). One of the eight ports of the gas manifold is located behind each tissue holder. Each rod is positioned immediately above a bottle (D), which contains the killing fluid. A piston housing with a cap (E) is located at the front top edge of the block. A smaller cylindrical channel (at the top right of the block (in Figs. 1 and 2) holds the WO, generator (F). The gas generator (F), the piston housing (E), and the
C
FIG. 4. Components of the apparatus: (A) Piston assembly (stainless steel) for delivery of acid for “CO, generation. (B) Teflon holder with trough for holding V-labeled barium carbonate. The piston assembly is inserted in the Teflon holder. (C) Combined leaf tissue holder, punch, and bottle seal. Eight of these are required. (D) Piston for moving “CO, from generating chamber to holding chamber and then to working chamber. Rod of stainless steel with piston of Teflon. (E) Legs of anodized aluminum.
254
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
FIG. 5. (A) Air inlet (s/s). (B) Components for needle valve for air inlet assembly for flushing chamber with nonradioactive air. (C) Stainless-steel cock (two required) for control of gas flow within block. (D) Stainless-steel end assembly for working chamber (two required). (E) Black-anodized aluminum bottle for killing and extraction medium (eight required). Combined vertical section (left) and exterior view (right).
working chamber (A) are interconnected by a system of channels, controlled by two cocks (G and H) , Further details of components are given in Figs. 3-5. A small thermometer is housed permanently in a slot cut into the back wall of the working chamber. Construction of apparatus. The following notes are intended for guidance on particular points during construction of the apparatus. Cut Perspex block. Machine all sides but leave outside dimensions 1/1s” larger than in Fig. 3. Anneal Perspex block according to manufacturer’s specifications. Polish block lightly and bore the working chamber in the lathe to approximately s/4” diameter. Mark out the 8 vertical holes; drill and ream through the total width of the block I/“. Counterbore the top of the block to the required dimensions and tap 7/116”26TPI in the lathe. Then mount tap in a chuck. Push the block with the tailstock center centered at the opposite side to the I/” bore. Tap carefully by rot,ating the chuck by hand. Drill the bottom holes out with a backedoff drill and tap 94” Unified National Fine using the above method. Counterbore the eight small chambers with a l/a” < +&” counterbore, at the top of the working chamber. Mount the block in the lathe and drill the gas manifold. Drill piston chamber and gas generating chamber with a multifluted drill or D bit. Do not predrill holes. Also drill the s2” connecting holes. Take block out of lathe and center the eight s2” cross gas channels with a sharp-pointed parallel engraving cutter or similar tool at such an angle that the drilled holes will line up with the manifold. Drill all channels &“. Predrill the holes for the two cocks 3/ls” and ream out to a 3” taper. Place block in lathe and machine the working chamber for half the
APPARATUS
FOR
CARBON
FIXATION
255
length of the block to 1” id. Make a 1” expanding arbor, mount block, and machine the rest of the working chamber. Provide threads for the end caps. To link up the two manifold channels, mill out a hole to a depth that will breach the channels in the middle. In turn seal this cavity with hexagon stainless steel cap and O-ring. Finish all other work and polish inside. Clean block with Teepol (detergent). On no account at this stage use organic solvents. Machine the block to the required dimensions. Polish and clean. Anneal the block again. To check any stresses use Polaroid glass and a light source for viewing. To make the tissue holders, use x6” stainless steel and drill out to 3/s” diameter and finish off with a 0.4134” end mill to a depth of 0.905”. The wall will be now 0.0138” thick. Machine the sharp edge. Tap the other side l/s” and part off. Fill the cutters with lead and mill half of the wall out. Remove lead and polish. METHOD
FOR USING
(A) Pre-equilibration
THE
APPARATUS
of Leaf Tissue
To ensure that the leaf sections are at maximal turgor during the WO, labeling, a period of equilibration in water-saturated conditions is necessary. 1. The tissue holders are removed from the apparatus and, as sections are excised from the leaf with a rectangular 2 cm X 1 cm cutter, they are placed on a holder. This is done using pointed forceps to hold the cut section and the thumb to push them gently onto the hooks. 2. The leaf holder is then placed on the apparatus and the bottom end of the section is dipped in water contained in a partially hollowed neoprene bung. The hollowed-out bung fits over the end of the tissue holder and seals the bottle holes under the working chamber. 3. The apparatus containing the leaf sections is then placed in a temperature-controlled room with the leaf sections under the desired illumination. The lamp is held horizontal so that the leaf surfaces are perpendicular to the incident light. Appropriate heat shielding of the apparatus is required. This is achieved by means of a sheet of plate glass placed between the lamp and the apparatus. In addition a current of air is blown across the lamp by means of a fan. 4. A water-saturated air flow is then passed through the chamber via the working chamber taps. These equilibration conditions are maintained over the time required for maximal turgor to be reached. This time was determined by cutting sets of leaf segments, floating them on water, and weighing batches at intervals. For Chinese cabbage leaf, increase in
256
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
weight ceased after 3-4 hr under these conditions. in the working chamber. (B)
Prepulse
A similar
time applied
Operations
1. The sample bottles are filled with 2.5 ml of methanol/chloroform/ water medium (h!lCW) (10,ll). 2. The sample bottles are then sealed with plastic self-sealing food wrap (Union Carbide). The plastic seal is held in place using an O-ring which fits in the top groove of the sample bottle. The sample bottles are then placed in chipped ice. A good seal is indicated when the surface of the plastic film becomes concave. 3. The neoprene bungs are removed from the apparatus and replaced by the sealed sample bottles. The tissue holders must be in their uppermost position before the bottles are screwed in. 4. Ba14C0, (1 mg) is then weighed into the Teflon container, which is then carefully pushed into the generating chamber. 5. The water-saturated air source is turned off and all taps, cocks, and valves are placed in the closed position. 6. The apparatus (still exposed to the light) is placed in an ethanol/ dry ice bath, the level of which should reach the top of the conduction rings of the sample bottles. (C) Labeling
with
14C0,
1. With cock G (Fig. 2) closed and cock H open the main piston IS drawn out about 3”. (This removes about 15 ml of air from the working chamber, which is used to give added preliminary dilution and mixing of the 14C0,.) Cock H is closed and G opened. 10% TCA (~0.5 ml) is then liberated from the generator (F of Fig. 2). The 14C0, is then flushed into the piston chamber by opening the needle valve and simultaneously drawing back the main piston about a further 1.5”. To prevent back-diffusion of ‘“COz, the needle valve and cock G are then closed while the main piston is still being withdrawn. (Two operators are needed at this stage.) 2. At time zero, the working chamber cock (H, Fig. 2) is open and the main piston rapidly compressed. The ‘“CO, now moves into the working chamber. The leaf sections are killed at the required times by pushing the tissue holder rods into their lowermost position. (D)
Completion
of the Experiment
1. After all the samples have been killed, the remaining “COz is flushed from the working chamber by a flow of air. The leaf holder rods are briefly withdrawn to the upper position to allow removal of any
APPARATUS
FOR
CARBON
FIXATION
257
gaseous ‘*CO, trapped in the sample bottles. This operation is conducted while the apparatus is still in the dry ice bath. 2. The whole apparatus is then placed in a 4” cold room for about 5 min to allow any ice that has formed around the threads of the bottles to melt. The bottles are removed; the killed leaf sections are removed from the hooks and placed back in their appropriate bottles, which are subsequently corked. 3. The Teflon Ba%O, container and the small piston are removed from the block and washed while the rest of the apparatus is flushed with water to ensure that no TCA is left in contact with the Perspex. (E)
Extraction
of Tissue
1. The leaf sections are left in MCW for 24 hr and then transferred to 2 ml of methanol/chloroform/formic acid (MCF) (10). The leaf section is ground in MCF. 2. The aqueous phase of the MCW and MCF extraction is obtained by the addition of 0.5 ml chloroform and 0.7 ml HZ0 per 2 ml of MCW or MCF and each pair pooled. 3. The aqueous extracts are taken to dryness using a rotary film evaporator and brought up to 0.3 ml in distilled water. DISCUSSION
AND
RESULTS
In work to be published elsewhere we have used the apparatus to make a detailed study of the effects of plant virus infection on the path of carbon fixation in photosynthesis. Here we summarize three experiments to indicate the general capabilities of the apparatus for brief labeling experiments with leaf tissue. The experiment summarized in Fig. 6 gives an indication of the variability to be expected in total fixation for different 2 X 1 cm pieces of tissue from the same leaf when equilibrated for 3.5 hr or when not so equilibrated. The data for positions 7, 5, 3, and 1 show that there is a rapid drop in the capacity of freshly excised tissue to fix carbon. There is a partial recovery after 3.5 hr (positions 2, 4, 6, 8). The experiment summarized in Fig. 7 shows the 4 set time courses for total fixation of “CO, by leaf from corn and Chinese cabbage under identical conditions. For our work, manual operation of the instrument has proved adequate. For greater accuracy at short time the operations could readily be mechanized. It is impossible to measure “killing time” precisely. It take a fraction of a second to move a leaf section from the working chamber to the bottle containing MCF at -72”. We chose the cold killing and extrac-
258
BEDBROOK,
VAN
SLUIS,
Position
AND
MATTHEWS
in chamber
FIG. 6. Effect of pre-equilibration of tissue, and holder position, on fixation of “CO1 by Chinese cabbage leaf tissue. Blocks of tissue (all taken from the same leaf) in positions 2, 4, 6, and 8 were pre-equilibrated in the apparatus for 3.5 hr at 2666 lumens/sq ft as described in the text. The other pieces of tissue were exercised at the following approximate times before the working chamber was closed and brought to saturated humidity again, just before the labeling period: (7) 0.5 min, (5) 1.5 min, (3) 2.5 min, (1) 3.5 min. Experiment carried out with plants grown in summer.
tion procedure since it has been shown to be much more effective than hot killing and extraction in minimizing post mortem enzyme activity (11)) and since it would be much more difficult to design an apparatus of the present type based on extraction with boiling solvents. For pulse-chase experiments the working chamber can be flushed rapidly with nonradioactive compressed air, without disturbing the leaf samples on the holders, To test the efficiency of flushing we used an air flow of about 10 times the volume of the working chamber per second (1 liter/set). We sampled the gas in the working chamber with a hypodermic syringe piercing a rubber stopper placed over tap B (right hand in Fig. 2). After a chase of 1 set, 90% of the 14C0, was removed. After 3 set 99.2% was removed. Removal of 99% of the radioactivity within 3 set is quite adequate for effective pulse-chase experiments. However we attempted to improve the efficiency of chasing by designing aerodynamically smooth inlets and outlets. In tests under the conditions outlined above and with an air flow of about 0.2 liter/see no improvement was obtained over the standard end assemblies shown in Fig. 5D. Used as we have outlined above, the introduction of ‘“CO, into the
APPARATUS
FOR
CARBON
Seconds
FIXATION
259
in 14C0 2
FIG. 7. Test of apparatus for time course experiments. Pieces of corn leaf tissue (0) were placed on four holders, and Chinese cabbage (0) on four. All pieces were pre-equilibrated for 3.5 hr at 2000 lumens/sq ft. For 20 min prior to the labeling period the chamber was flushed with water-saturated CO?-free air. “CO, was introduced into the working chamber and two pieces of tissue wrre killed at the times indicated. Experiment carried out with plants grown in winter.
working chamber raises the CO, concentration by about S-fold, aa calculated from the weight of BaC03 used and the volume of the chamber. By use of the appropriate taps, gas mixtures of any desired compo.Gtion can be introduced rapidly into the working chamber from an outside source. In our experiments we have used a single 400 W lamp at a distance to give about 2000 1umensJsq ft at the leaf surface. The use of higher light intensities would require the use of efficient heat filters between the light source and the leaf. We have maintained constant-temperature conditions within the working chamber from experiment to experiment by using the apparatus in a constant temperature and light room. Where variable-temperature conditions were required the working chamber could be modified to include heating, cooling, and control components. We have found that the eight chamber version of the apparatus using
260
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
2 X 1 cm pieces of leaf is adequate for many purposes. If required, the number of chambers could readily be increased up to about twelve or more. Where the use of excised leaf sections is considered unsatisfactory, the size of the working chamber, tissue holders, and bottles could be increased to accommodate small intact seedling plants. SUMMARY
An apparatus is described which allows kinetic studies to be made of photosynthetic carbon fixation using pieces of leaf tissue. Eight pieces of tissue 2 cm X 1 cm can be exposed to ‘“CO, under identical conditions for periods down to about 1 sec. 90% of the ‘“CO, can be flushed from the chamber in 1 set, and about 99% in 3 sec. ACKNOWLEDGMENT We wish to thank Professor R. F. Meyer of the School of Engineering, of Auckland, for advice on the design of end assemblies.
University
REFERENCES 1. BASSHAM, 2. 3. 4. 5.
6. 7. 8. 9.
10. 11.
J. A., BENSON, A. A., KAY, L. D., HARRIS, A. Z., WILSON, A. T., AND CALVIN, M., J. Amer. Chem. Sot. 76, 1760 (1954). KORTSCHAK, H. P., HORTT, C. E., AND BURR, G. O., Plant Physiol. 40, 209 (1965). HULL, R. J., AND LEONARD, 0. A., Plant Physiol. 39, 996 (1964). BIGGINS, J., AND PARK, R. B., Plant Physiol. 41, 115 (1966). HOMANN, P. H., Plant Physiol. 42, 997 (1967). ADAMS, M. S., STRAIN, B. R., AND TING, I. P., Plant Physiol. 42, 1797 (1967). ABRAHAMSEN, M., AND MAYER, A. M., Physiol. Plantarum 29, 1 (1967). HATCH, M. D., AND SLACK, C. R., Biochem. J. 101, 103 (1966). HATCH, M. D., SLACK, C. R., AND JOHNSON, H. S., Biochem. J. 417, (1967). BIELESKI, R. L.. AND YOUNG, R. E.. Anal. Biochem. 6, 54 (1963). BIELESKI, R. L., And. Biochem. 9, 431 (1964).
46,
BIOCHEMISTRY
Apparatus
for
250-260
(1972)
Kinetic
Studies
in Leaf J. R. BEDBROOK, Department
of Cell
D.
Biology,
VAN
Received
AND
R. E. F. MATTHEWS
of Auckland, July
Fixation
Tissue
SLUIS,
University
of Carbon
Auckland,
New
Zealand
28, 1971
The most detailed studies on the path of carbon fixation in phot.osynthesis have been carried out with algae, where flow methods can conveniently be applied (1). Carbon dioxide fixation in leaves has usually been studied using labeling periods of minutes or hours, and relatively simple devices for exposure of the tissue to CO, (2-7). Hatch and Slack (8) and Hatch et al. (9) used a simple Perspex box which could accommodate six leaf segments. Individual segments could be removed from the box at different times. We recently began to study the effects of virus infection on the path of carbon fixation in leaf tissue. For this work we needed to be able to expose small pieces of healthy and virus-infected leaf to ‘“CO, under identical conditions for a range of short labeling times, We describe here an apparatus which allows eight pieces of leaf tissue to be exposed to ‘“COz in a single working chamber. The ‘“COz is generated in a subsidiary chamber and transferred to a holding chamber by means of a piston. At the beginning of the labeling period the piston is used to force the ‘“CO, rapidly and uniformly through a manifold into the working chamber. The labeling period for each sample can be varied independently down t.o about one second. MATERIALS
AND
METHODS
General. Chinese cabbage (Brassica pekinensis Rupr.) var. Wong Bok and corn (Zea mays L.) were grown in the glasshouse under conditions of natural lighting. A rectangular block of brass was milled to make a cutter 2 cm X 1 cm, which was used to take leaf samples of uniform area. For labeling experiments leaf samples held at a temperature of 25-27” were exposed to light of 2000-2200 lumensjsq ft from a Philips HLRG 400 W horticultural lamp. l”COz was generated from 14C-labeled barium carbonate (CFA 431, Radiochemical Centre, Amersham, England). Description of apparatus. The apparatus is illustrated in Fig. 3 and 250 @ 1972 by
Academic
Press,
Inc.
APPARATCS
FIG.
FOR
1. General
CARRON
view
251
FIXATION
of the
assembly.
nL .=
L1 FIG. 2. Front see text.
elevation
of
apparatus
showing
E
completed
assembly.
For
details
252
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
FIG. 3. Working drawings showing drilling of Perspex block. Two vertical sections of the block are shown. The upper vertical section is drawn through the center of the working chamber. The lower vertical section is drawn through the center of the ‘“CO, generating chamber and shows the piston and manifold for distributing the gas to the working chamber.
shown in front elevation in Fig. 2. A solid block of Perspex (poly(methy1 methacrylate), Imperial Chemical Industries Ltd.) is machined and bored to form a working chamber (A), which is closed at both ends with a stainless-steel cap (B). The caps are provided with gate taps. The working chamber is pierced vertically with eight channels which in turn contain eight tissue holders (numbered l-8 in Fig. 2). These are sealed with a cap (C) and an O-ring. The O-ring also acts as a friction brake. The piece of tissue is held in place by three metal hooks (shown
APPARATUS
FOR
CARBON
FIXATION
253
in Fig. 4C). One of the eight ports of the gas manifold is located behind each tissue holder. Each rod is positioned immediately above a bottle (D), which contains the killing fluid. A piston housing with a cap (E) is located at the front top edge of the block. A smaller cylindrical channel (at the top right of the block (in Figs. 1 and 2) holds the WO, generator (F). The gas generator (F), the piston housing (E), and the
C
FIG. 4. Components of the apparatus: (A) Piston assembly (stainless steel) for delivery of acid for “CO, generation. (B) Teflon holder with trough for holding V-labeled barium carbonate. The piston assembly is inserted in the Teflon holder. (C) Combined leaf tissue holder, punch, and bottle seal. Eight of these are required. (D) Piston for moving “CO, from generating chamber to holding chamber and then to working chamber. Rod of stainless steel with piston of Teflon. (E) Legs of anodized aluminum.
254
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
FIG. 5. (A) Air inlet (s/s). (B) Components for needle valve for air inlet assembly for flushing chamber with nonradioactive air. (C) Stainless-steel cock (two required) for control of gas flow within block. (D) Stainless-steel end assembly for working chamber (two required). (E) Black-anodized aluminum bottle for killing and extraction medium (eight required). Combined vertical section (left) and exterior view (right).
working chamber (A) are interconnected by a system of channels, controlled by two cocks (G and H) , Further details of components are given in Figs. 3-5. A small thermometer is housed permanently in a slot cut into the back wall of the working chamber. Construction of apparatus. The following notes are intended for guidance on particular points during construction of the apparatus. Cut Perspex block. Machine all sides but leave outside dimensions 1/1s” larger than in Fig. 3. Anneal Perspex block according to manufacturer’s specifications. Polish block lightly and bore the working chamber in the lathe to approximately s/4” diameter. Mark out the 8 vertical holes; drill and ream through the total width of the block I/“. Counterbore the top of the block to the required dimensions and tap 7/116”26TPI in the lathe. Then mount tap in a chuck. Push the block with the tailstock center centered at the opposite side to the I/” bore. Tap carefully by rot,ating the chuck by hand. Drill the bottom holes out with a backedoff drill and tap 94” Unified National Fine using the above method. Counterbore the eight small chambers with a l/a” < +&” counterbore, at the top of the working chamber. Mount the block in the lathe and drill the gas manifold. Drill piston chamber and gas generating chamber with a multifluted drill or D bit. Do not predrill holes. Also drill the s2” connecting holes. Take block out of lathe and center the eight s2” cross gas channels with a sharp-pointed parallel engraving cutter or similar tool at such an angle that the drilled holes will line up with the manifold. Drill all channels &“. Predrill the holes for the two cocks 3/ls” and ream out to a 3” taper. Place block in lathe and machine the working chamber for half the
APPARATUS
FOR
CARBON
FIXATION
255
length of the block to 1” id. Make a 1” expanding arbor, mount block, and machine the rest of the working chamber. Provide threads for the end caps. To link up the two manifold channels, mill out a hole to a depth that will breach the channels in the middle. In turn seal this cavity with hexagon stainless steel cap and O-ring. Finish all other work and polish inside. Clean block with Teepol (detergent). On no account at this stage use organic solvents. Machine the block to the required dimensions. Polish and clean. Anneal the block again. To check any stresses use Polaroid glass and a light source for viewing. To make the tissue holders, use x6” stainless steel and drill out to 3/s” diameter and finish off with a 0.4134” end mill to a depth of 0.905”. The wall will be now 0.0138” thick. Machine the sharp edge. Tap the other side l/s” and part off. Fill the cutters with lead and mill half of the wall out. Remove lead and polish. METHOD
FOR USING
(A) Pre-equilibration
THE
APPARATUS
of Leaf Tissue
To ensure that the leaf sections are at maximal turgor during the WO, labeling, a period of equilibration in water-saturated conditions is necessary. 1. The tissue holders are removed from the apparatus and, as sections are excised from the leaf with a rectangular 2 cm X 1 cm cutter, they are placed on a holder. This is done using pointed forceps to hold the cut section and the thumb to push them gently onto the hooks. 2. The leaf holder is then placed on the apparatus and the bottom end of the section is dipped in water contained in a partially hollowed neoprene bung. The hollowed-out bung fits over the end of the tissue holder and seals the bottle holes under the working chamber. 3. The apparatus containing the leaf sections is then placed in a temperature-controlled room with the leaf sections under the desired illumination. The lamp is held horizontal so that the leaf surfaces are perpendicular to the incident light. Appropriate heat shielding of the apparatus is required. This is achieved by means of a sheet of plate glass placed between the lamp and the apparatus. In addition a current of air is blown across the lamp by means of a fan. 4. A water-saturated air flow is then passed through the chamber via the working chamber taps. These equilibration conditions are maintained over the time required for maximal turgor to be reached. This time was determined by cutting sets of leaf segments, floating them on water, and weighing batches at intervals. For Chinese cabbage leaf, increase in
256
BEDBROOK,
VAN
SLUIS,
AND
MATTHEWS
weight ceased after 3-4 hr under these conditions. in the working chamber. (B)
Prepulse
A similar
time applied
Operations
1. The sample bottles are filled with 2.5 ml of methanol/chloroform/ water medium (h!lCW) (10,ll). 2. The sample bottles are then sealed with plastic self-sealing food wrap (Union Carbide). The plastic seal is held in place using an O-ring which fits in the top groove of the sample bottle. The sample bottles are then placed in chipped ice. A good seal is indicated when the surface of the plastic film becomes concave. 3. The neoprene bungs are removed from the apparatus and replaced by the sealed sample bottles. The tissue holders must be in their uppermost position before the bottles are screwed in. 4. Ba14C0, (1 mg) is then weighed into the Teflon container, which is then carefully pushed into the generating chamber. 5. The water-saturated air source is turned off and all taps, cocks, and valves are placed in the closed position. 6. The apparatus (still exposed to the light) is placed in an ethanol/ dry ice bath, the level of which should reach the top of the conduction rings of the sample bottles. (C) Labeling
with
14C0,
1. With cock G (Fig. 2) closed and cock H open the main piston IS drawn out about 3”. (This removes about 15 ml of air from the working chamber, which is used to give added preliminary dilution and mixing of the 14C0,.) Cock H is closed and G opened. 10% TCA (~0.5 ml) is then liberated from the generator (F of Fig. 2). The 14C0, is then flushed into the piston chamber by opening the needle valve and simultaneously drawing back the main piston about a further 1.5”. To prevent back-diffusion of ‘“COz, the needle valve and cock G are then closed while the main piston is still being withdrawn. (Two operators are needed at this stage.) 2. At time zero, the working chamber cock (H, Fig. 2) is open and the main piston rapidly compressed. The ‘“CO, now moves into the working chamber. The leaf sections are killed at the required times by pushing the tissue holder rods into their lowermost position. (D)
Completion
of the Experiment
1. After all the samples have been killed, the remaining “COz is flushed from the working chamber by a flow of air. The leaf holder rods are briefly withdrawn to the upper position to allow removal of any
APPARATUS
FOR
CARBON
FIXATION
257
gaseous ‘*CO, trapped in the sample bottles. This operation is conducted while the apparatus is still in the dry ice bath. 2. The whole apparatus is then placed in a 4” cold room for about 5 min to allow any ice that has formed around the threads of the bottles to melt. The bottles are removed; the killed leaf sections are removed from the hooks and placed back in their appropriate bottles, which are subsequently corked. 3. The Teflon Ba%O, container and the small piston are removed from the block and washed while the rest of the apparatus is flushed with water to ensure that no TCA is left in contact with the Perspex. (E)
Extraction
of Tissue
1. The leaf sections are left in MCW for 24 hr and then transferred to 2 ml of methanol/chloroform/formic acid (MCF) (10). The leaf section is ground in MCF. 2. The aqueous phase of the MCW and MCF extraction is obtained by the addition of 0.5 ml chloroform and 0.7 ml HZ0 per 2 ml of MCW or MCF and each pair pooled. 3. The aqueous extracts are taken to dryness using a rotary film evaporator and brought up to 0.3 ml in distilled water. DISCUSSION
AND
RESULTS
In work to be published elsewhere we have used the apparatus to make a detailed study of the effects of plant virus infection on the path of carbon fixation in photosynthesis. Here we summarize three experiments to indicate the general capabilities of the apparatus for brief labeling experiments with leaf tissue. The experiment summarized in Fig. 6 gives an indication of the variability to be expected in total fixation for different 2 X 1 cm pieces of tissue from the same leaf when equilibrated for 3.5 hr or when not so equilibrated. The data for positions 7, 5, 3, and 1 show that there is a rapid drop in the capacity of freshly excised tissue to fix carbon. There is a partial recovery after 3.5 hr (positions 2, 4, 6, 8). The experiment summarized in Fig. 7 shows the 4 set time courses for total fixation of “CO, by leaf from corn and Chinese cabbage under identical conditions. For our work, manual operation of the instrument has proved adequate. For greater accuracy at short time the operations could readily be mechanized. It is impossible to measure “killing time” precisely. It take a fraction of a second to move a leaf section from the working chamber to the bottle containing MCF at -72”. We chose the cold killing and extrac-
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FIG. 6. Effect of pre-equilibration of tissue, and holder position, on fixation of “CO1 by Chinese cabbage leaf tissue. Blocks of tissue (all taken from the same leaf) in positions 2, 4, 6, and 8 were pre-equilibrated in the apparatus for 3.5 hr at 2666 lumens/sq ft as described in the text. The other pieces of tissue were exercised at the following approximate times before the working chamber was closed and brought to saturated humidity again, just before the labeling period: (7) 0.5 min, (5) 1.5 min, (3) 2.5 min, (1) 3.5 min. Experiment carried out with plants grown in summer.
tion procedure since it has been shown to be much more effective than hot killing and extraction in minimizing post mortem enzyme activity (11)) and since it would be much more difficult to design an apparatus of the present type based on extraction with boiling solvents. For pulse-chase experiments the working chamber can be flushed rapidly with nonradioactive compressed air, without disturbing the leaf samples on the holders, To test the efficiency of flushing we used an air flow of about 10 times the volume of the working chamber per second (1 liter/set). We sampled the gas in the working chamber with a hypodermic syringe piercing a rubber stopper placed over tap B (right hand in Fig. 2). After a chase of 1 set, 90% of the 14C0, was removed. After 3 set 99.2% was removed. Removal of 99% of the radioactivity within 3 set is quite adequate for effective pulse-chase experiments. However we attempted to improve the efficiency of chasing by designing aerodynamically smooth inlets and outlets. In tests under the conditions outlined above and with an air flow of about 0.2 liter/see no improvement was obtained over the standard end assemblies shown in Fig. 5D. Used as we have outlined above, the introduction of ‘“CO, into the
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FIG. 7. Test of apparatus for time course experiments. Pieces of corn leaf tissue (0) were placed on four holders, and Chinese cabbage (0) on four. All pieces were pre-equilibrated for 3.5 hr at 2000 lumens/sq ft. For 20 min prior to the labeling period the chamber was flushed with water-saturated CO?-free air. “CO, was introduced into the working chamber and two pieces of tissue wrre killed at the times indicated. Experiment carried out with plants grown in winter.
working chamber raises the CO, concentration by about S-fold, aa calculated from the weight of BaC03 used and the volume of the chamber. By use of the appropriate taps, gas mixtures of any desired compo.Gtion can be introduced rapidly into the working chamber from an outside source. In our experiments we have used a single 400 W lamp at a distance to give about 2000 1umensJsq ft at the leaf surface. The use of higher light intensities would require the use of efficient heat filters between the light source and the leaf. We have maintained constant-temperature conditions within the working chamber from experiment to experiment by using the apparatus in a constant temperature and light room. Where variable-temperature conditions were required the working chamber could be modified to include heating, cooling, and control components. We have found that the eight chamber version of the apparatus using
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2 X 1 cm pieces of leaf is adequate for many purposes. If required, the number of chambers could readily be increased up to about twelve or more. Where the use of excised leaf sections is considered unsatisfactory, the size of the working chamber, tissue holders, and bottles could be increased to accommodate small intact seedling plants. SUMMARY
An apparatus is described which allows kinetic studies to be made of photosynthetic carbon fixation using pieces of leaf tissue. Eight pieces of tissue 2 cm X 1 cm can be exposed to ‘“CO, under identical conditions for periods down to about 1 sec. 90% of the ‘“CO, can be flushed from the chamber in 1 set, and about 99% in 3 sec. ACKNOWLEDGMENT We wish to thank Professor R. F. Meyer of the School of Engineering, of Auckland, for advice on the design of end assemblies.
University
REFERENCES 1. BASSHAM, 2. 3. 4. 5.
6. 7. 8. 9.
10. 11.
J. A., BENSON, A. A., KAY, L. D., HARRIS, A. Z., WILSON, A. T., AND CALVIN, M., J. Amer. Chem. Sot. 76, 1760 (1954). KORTSCHAK, H. P., HORTT, C. E., AND BURR, G. O., Plant Physiol. 40, 209 (1965). HULL, R. J., AND LEONARD, 0. A., Plant Physiol. 39, 996 (1964). BIGGINS, J., AND PARK, R. B., Plant Physiol. 41, 115 (1966). HOMANN, P. H., Plant Physiol. 42, 997 (1967). ADAMS, M. S., STRAIN, B. R., AND TING, I. P., Plant Physiol. 42, 1797 (1967). ABRAHAMSEN, M., AND MAYER, A. M., Physiol. Plantarum 29, 1 (1967). HATCH, M. D., AND SLACK, C. R., Biochem. J. 101, 103 (1966). HATCH, M. D., SLACK, C. R., AND JOHNSON, H. S., Biochem. J. 417, (1967). BIELESKI, R. L.. AND YOUNG, R. E.. Anal. Biochem. 6, 54 (1963). BIELESKI, R. L., And. Biochem. 9, 431 (1964).