isolation mesophyll cells from telephium · pdf filemncl2, 2 mmnano.,, 5 mmmgcl2. 5 mm ... the...

Plant Physiol. (1973) 51, 97-103

Isolation of Mesophyll Cells from Sedum telephium Leaves1Received for publication July 17, 1972

I. ROUHANI AND H. M. VINESDepartment of Horticulture, University of Georgia, Athens, Georgia 30601

C. C. BLACK, Jr.Department of Biochemistry, University of Georgia, Athens, Georgia 30601

ABSTRACT

A technique is described for mechanically isolating metabol-ically active individual spongy mesophyll cells from the Cras-sulacean acid metabolism plant, Sedum telephium. Matureleaves are selected at about 2 PM when acidity is low, and threedifferent media are used to reduce the problem of leaf acidityand to maintain isotonic conditions. The upper and lower epi-dermis is peeled from chilled leaves and the leaves are sus-pended in a buffered "soaking medium," then gently groundwith a mortar and pestle. Cells and debris are separated using a"washing medium," with cells being filtered through a 136micron net and collected on an 80 micron net. Cells then aresuspended in a "cell suspension medium" and concentrated bycentrifugation. Approximately 2 hours are required for theisolation procedure, and activity in C02 fixation is constant forup to 4 hours after isolation. Microscopic examination showsabout 65% of the isolated cells appear intact and unplasmo-lyzed and are similar to leaf msophyll cells. The yield of cellson a leaf chlorophyll basis is about 1%. A light micrograph ofthe isolated cells is given.The isolated cells upon addition of phosphoenolpyruvate, 2-

phosphoglycerate, and ribulose-1, 5-diphosphate fix C02 asrapidly as intact leaves; however, without exogenous substratethe cells only fix C02 between 10 and 20% of intact leaves. Thetemperature and pH optima for cellular C02 fixation in thepresence of phosphoenolpyruvate is 35 to 45 C and 7.5 to 9.0,respectively. The light and dark portions of C02 fixation withthe isolated cells are considered in relation to a scheme for netC02 fixation by Crassulacean acid metabolism plants.

Crassulacean acid metabolism plants generally are charac-terized by the accumulation of titratable acidity at night, fol-lowed by a loss of acidity during the day in their leaves, whilethe leaf stomata open principally at night, and starch accumu-lation and degradation occur principally in the day and night,respectively (2, 5, 16). The CAM2 plants which have beenexamined possess an active PEP carboxylase (22, 23), whichpresumably is involved in the organic acid accumulation in

1 This research was supported in part by National Science Foun-dation Grant GB 7772.

2 Abbreviations: CAM: Crassulacean acid metabolism; PEP:phosphoenol-pyruvate; C4: C4-dicarboxylic acid; 3-PGA: 3-phospho-glycerate; 2-PGA: 2-phosphoglycerate; RuDP: ribulose-1, 5-diphos-phate; R-5-P: ribose-5-phosphate; 6-PGA: 6-phosphogluconate:OAA: oxaloacetate.

CAM plant leaves (3, 6, 18, 19). A renewal of interest in CAMplant metabolism has occurred recently due to the discovery ofthe C, cycle of photosynthetic carbon metabolism in sugarcaneand other plants (11, 14). The C4 cycle also employs PEP car-boxylase as a major leaf carboxylase (11, 12). CAM and C4plants have many other similarities, and the postulation hasbeen presented (9, 15) that CAM plants have a temporallyseparated acid formation and degradation occurring in eachleaf photosynthetic cell, whereas leaves of C4 plants have spa-tially separated acid formation, which occurs in mesophyll cellsfrom acid degradation occurring in bundle sheath cells.

This laboratory has been involved in cell isolation studies toresolve the metabolism of organs such as leaves (4, 10, 20, 21).This manuscript reports a technique for isolating leaf meso-phyll cells from a CAM plant, Sedum telephiumn, and presentssome metabolic studies with the isolated cells.

MATERIALS AND METHODS

Culturing Plants. A stock plant of Seduin telephium, grow-ing in Athens, Georgia was selected, and foliage cuttings wererooted then transplanted in a medium of soil, sand, and ver-miculite (1 :1:1 v/v/v) in a house with a light intensity between4000 and 6000 ft-c. Day temperatures ranged from 21 to 25C, and night temperatures ranged from 17 to 20 C. The plantswere watered two times per week in the morning.

`CO2 Incorporation with Isolated Cells. In addition to sub-strates, cells, and cofactors. as specified in each experimentalsetup, the total mixture of 250 ,ul contained: 50 mm Trizmabase adjusted with MES to pH 8.0, 2 mm EDTA, 1 mMMnCl2, 2 mm NaNO.,, 5 mm MgCl2. 5 mM K2HPO4, and 350mM sorbitol. The vial was placed in a 30 C temperature-con-trolled water bath. Light intensity was 2500 ft-c at the reactionmixture surface. Two minutes were allowed for temperatureadjustment, and then "4C-labeled NaH CO. was pipetted intothe mixture at a final concentration of 5 mm to start the re-action. Each vial was shaken a few times during the course ofthe experiments to prevent cell precipitation. Samples of 50p.1 were taken at specific times and put in a vial containing 50,ul of 20% (w/v) trichloroacetic acid. Scintillation liquid (11ml) was added. and unincorporated "CO2 removed by N. flush-ing for I min before the samples were counted for appropriatetimes. Liquid scintillation solutions were prepared by adding4 g of BBOT (2,5-tert-butyl-benzoxyzalythiophene) to 1 literof 700 ml of toluene plus 300 ml of ethanol solution and keptat 10 C in the dark (8. 17).

RESULTS

A number of CAM plant leaves were surveyed to see if in-tact photosynthetic cells could be released by mechanical meansusing a mortar and pestle. The plants examined included:

97

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ROUHANI, VINES, AND BLACK

GRIND IN MORTAR AND PESTLE

I IlOg of peeled S taluRblm leaves ore ground gently with a mortar andpestle in lOml of Sol. A.

FILTERlOml of Sol. B oar added to the homogenate, followed by filtration through a20mesh stainless steel sieve in a Falcon FlIltr Unit, while stirring with aglass rod. The mortar and pestle are washed with lOmI of Sol. B and pouredonto the sieve.

REGRIND UNMACERATED TISSUEUnmacerated tIue is removed from the sieve and ground gently, thenfiltrated as above. This step may be repeated 5 or 6 times. Filtrotes aresaved.

SIEVEAbout 20ml lots of filtrate are added to test tubes(24xl20mm) and shakenfor 30 seconds and fIltered through a 30 mesh stoins steel sie In a

Falcon Filter Unit while being stirred with aglass rod. Entire filtrate isshee before passing through slove.

SEPARATIONFiltrateis ogain shokeeas before for one minute, and paed tihugh a 136mic nylon nt above an 80micron nylon not in a PlexIglass filter urIt withslow magnetIc stirring, and then washed with 3 volunms of Sol B.

ICOLLECTION

Cells are collected on the \Cell clumps on the 136 micron80mlcon net and washed 3 net are suspended and shokentImes with Sol. B ogaIn, and filtered through the 136

micron net obove the 80 micron net.

CENTRI FUGATIONCel suspnsion is centrifuged at 750xg for one minute and thenresuspended by shaking in Sol C ( ml ) These isolated spongy mesophyllcells are stored In on Ice bucKet until needed for assay.

FIG. 1. Flow diagram for isolating S. telephium spongy meso-phyll cells. For the isolation of cells, three solutions are used. Solu-tion A (soaking medium) contained Trizma base (60 mM) adjustedwith MES buffer to pH 8.5, 5 mM MgC92, 5 mM K2HPO4, 2 mMNaNO3, 2 mm EDTA, and 1 mM MnCI2. Solution B (washing me-dium) contained 0.3 M sorbitol, 50 mM Trizma base adjusted withMES to pH 8.5, and 5 mM MgCl2. Solution C (cell suspension me-dium) contained 50 mM Trizma base adjusted with MES to pH 8.0,2 mm EDTA, 1 mm MnCl2, 2 mM NaNO3, 5 mM MgC12, 5 mMK)HP04, and 0.35 M sorbitol.

'For details on types of material and equipment used see ref. 10.2 The test tubes were shaken with a Deluxe Mixer, fast setting, at

room temperature.

Crassula argentea, Sedum telephoides, Seduin telephiutn, Bryo-phyllum crenata, Bryophyllum daigremontiana, Kalanchoetubiflora Hamet, and a cactus species. With Sedum telephiumnit seemed, from preliminary examinations after gently grindingthe leaves with a mortar and pestle, that whole cells werevisible in a light microscope and thus might be isolated. Fewwhole cells were visible in similar preparations from otherCAM plant leaves.

The S. telephium plants used for cell isolation were exposedto light of 2500 ft-c for at least 8 hr prior to isolation in orderto reduce the leaf acidity. Fully expanded leaves were removedfrom about three plants of similar size and placed on crushedice for about 30 min before being washed with distilled water.After blotting dry, 30 g of healthy leaves were selected, and themargin of each leaf was removed with a dissecting knife. Theleaves were bent slightly and both the upper and lower epider-mis were peeled off by hand, and the midribs were removed.From 30 g of whole leaf tissue, about 10 g of peeled leaf tissuecould be separated. The peeled leaf portions were soaked in20 ml of soaking medium (Fig. 1) and kept at 10 to 15 C untilall leaves were prepared. This portion of the preparationusually took 1 to 2 hr. All solutions were kept at ice bathtemperature, and the filtering and related operations in Fig-ure 1 were at room temperatures, 21 to 24 C.

The peeled leaf sections were ground "very" gently (see Fig.I for comparison) in 10 ml of solution A at 10 C using amortar and pestle as outlined in the flow diagram. Periodicchecks were made with a light microscope (Bausch and LombDynazoom, Model 31) to see the degree of cell breakage andcell separation during the course of following the procedureoutlined in Figure 1.The percentage of intact cells in several preparations was

calculated after counting the number of cells in a cell sample(50 1.d) with a light microscope. The data in Table I show thatthe percentage of intact cells was between 55 and 68%, and thepercentage broken or plasmolyzed cells was between 32 and45%.The yield of cells in various preparations on a chlorophyll

basis was determined by assaying for total leaf chlorophyll andfor total chlorophyll in the isolated cell preparations. The yieldof spongy mesophyll cells from S. telephiumn leaves was about1 % (Table II).A photograph of a typical light microscope field of isolated

spongy mesophyll cells from S. telephium leaves is shown inFigure 2. Light microscopic examinations during these investi-gations showed that the majority of cells isolated by this pro-cedure retained shapes similar to those observed in tissue sec-tions.

These isolated cells where active in fixing CO2 for at least 4hr after isolation when stored in an ice bath. In the microscope,they appear to be intact for periods up to 10 hr.A compilation of the ideas of previous workers with CAM

plants is given in Figure 3 (4) and our studies with isolatedcells were designed to test the validity of the scheme in singleleaf photosynthetic cells.

Table I. Cell Countts in Typical Preparationis of Spongy MesophyllCells Isolated from Sedum telephiunt Leaves

Experiment

I

2

3

4

Intact and Broken CellsCollected on the 80 p Net

Total Cells Counted

Intact Broken orplasmolyzed

2307 61 392812 58 422771 66 343047 60 403800 68 323550 55 454000 63 374100 68 32

Table II. Typical Yields oii a Chlorophyll Basis of SponigyMesophyll Cells Isolated from Sedum telephium Leaves

Experiment Preparation' Total Chlorophyll Yield

mg N1 Leaf 4.09 100

Cells 0.036 0.92 i Leaf 3.39 100

Cells 0.030 0.93 Leaf 3.60 100

Cells 0.037 14 Leaf 4.00 100

Cells 0.040 1

In these experiments 6 g of peeled leaf tissue was used.

98 Plant Physiol. Vol. 51, 1973



Plant Physiol. Vol. 51, 1973

FIG. 2. Light micrograph of spongy mesophyll cells isolated from S. telephiutm leaves. Arrow A shows a broken and B shows a plasmolyzedcell. X 200.

The cells as isolated were active in fixing CO2 in light andin dark (Table III and Fig. 4). This endogenous activityvaried in daily experiments from about 3 to 14 ,umoles of CO2fixed/mg chl hr, and the effect of light varied from no effectto about a 50% stimulation.

Feeding PEP to isolated cells strongly stimulated CO2 fixa-tion in both light and dark (Fig. 4 and Table III). Since CO2fixation occurred at a substantial rate in this reaction mixture,it was investigated in some detail to characterize more fullythe isolated cells and to delineate the optimal experimentalconditions for maximal CO2 fixation activity. Initial linearityof CO2 fixation with time and with chlorophyll (or cell) con-centration was observed (Figs. 4 and 5). A fairly broad pHoptimum between 7.5 and 9.0 was observed (Fig. 6). The cellsresponded markedly to temperature (Fig. 7) with a broadoptimum near 35 to 45 C (E. = 11.48 kcal/mole).The influence of PEP concentrations on the CO, fixation is

shown in Figure 8 with a Km value of about 0.7 mm and Vmaxat about 10 mM PEP. These experiments also were conductedin both light and dark with similar rates being observed (TableIII). 2-Phosphoglyceric acid fed to cells at various concentra-tions was almost as effective as PEP in supporting CO, fixation(Table III). A Km for 2-PGA of about 0.9 mm and Vmax of 5mM occurred. A stimulation of CO2 fixation was observed with3-PGA; however, the rate was only about one-tenth of thePEP rate. An apparent Km of 0.2 mm and a V.a. of about 2.5mm was observed.

R-5-P effectively promotes CO2 fixation particularly if theproper ratio of MgCI2 to R-5-P is achieved (about 5: 1) (TableIII and Fig. 9). RuDP also was quite effective in promoting

3

3 G-3-PF-i~~~~fF-1 ,6-DP 3 fDA

3 DHAP6

+ PP

CO2FIXATION IN CRASSULACEAN PLANTS IN W5 \W,!)DARK (A) IN LIGHT ( A & B ).

FIG. 3. Scheme for net CO2 fixation in Crassulacean acid me-tabolism plants in dark (A) and in light (A and B).

SEDUM LEAF CELLS 99

11p NADPH

.i




Table III. Summary of CO2 Fixationi by Isolated Sedum telephiumMesophyll Cells under Various Experimenztal Conditions

Experi- 1Ratement Treatment and Compounds Added of C02

Fixation

Jgmoles/mgchi * hr

1 Light, no addition 6.6Dark, no addition 4.5

2 Light, boiled cells, PEP (5.0 mM) 1.7Light, unboiled cells, PEP (5.0 mM) 127.0

3 Light, PEP (2.5 mM) 125.0Dark, PEP (2.5 mM) 82.0Light, PEP (5.0 mM) 132.0Dark, PEP (5.0 mM) 128.0Light, PEP (8.0 mM) 142.0Dark, PEP (8.0 mM) 134.0

4 Light, 3-PGA (0.4 mM) 40.0Dark, 3-PGA (0.4 mM) 48.0

5 Light, 6-PGA (0.2 mM) 32.0Dark, 6-PGA (0.2 mM) 35.0

6 Light, RuDP (0.6 mM) 62.0Dark, RuDP (0.6 mM) 58.0

7 Light, pyruvate (2.0 mM) 7.5Light, ATP (2.0 mM) 8.7Dark, ATP (2.0 mM) 3.5Light, pyruvate (3.0 mM), ATP (1.0 mm) 27.2Light, R-5-P (5.0 mM), ATP (1.0 mM) 28.0Dark, R-5-P (5.0 mM), ATP (1.0 mM) 18.0

CO2 fixation as were some other compounds, and their influ-ences on CO2 fixation are noted (Table III).

DISCUSSION

In the complete cell isolation procedure, three kinds ofmedia were used. CAM plant leaf cells contain high amountsof acid presumed to be in the vacuole (6); when the leaveswere ground, a low pH (4-6) often was observed which may

inactivate organelles and enzymes. So the first medium (solu-tion A) has a high pH (8.5), and the leaves were soaked in itabout 1 hr. During that time, the medium presumably pene-

trated inside the cells, thereby reducing the acidity, althoughthis was not actually tested. Solution A also did not containany additional osmoticum such as sugars. which might allowbetter penetration of the buffer. The epidermis was removedto reduce the amount of waxes and phenolic components andto allow a better penetration of the medium. When the leaveswere ground, 0.35 M sorbitol was added to give an isotonicosmotic potential near that of most plant tissues to prevent cel-lular collapse or plasmolysis. Usually after grinding the leaves,the pH of the suspension was about 7.0 to 7.5. The cells finallywere suspended in a solution which maintained the osmoticpotential, but which had a lower buffer capacity so that one

could more easily study cell metabolism, e.g., it would beeasier to var) pH.One of tht problems encountered in isolating cells from

S. telephium was that the cells packed together when the leaveswere ground to mechanically separate the cells. The reason forthis may be that there is some attractive force between them,

but it was clear that the cells were sticky to the touch. To over-

come this problem, the technique described in Figure 1 hasseveral shaking steps in dilute medium prior to the filtrationsteps. Even after isolation, the cells needed to be shaken im-mediately prior to selecting a sample.The spongy mesophyll cells of S. telephiumn, when compared

with mesophyll cells of crabgrass and spinach (10), are muchlarger in size. Spongy mesophyll cells of S. telephium have a

diameter between 80 and 136 ,u, while the diameter of crab-grass mesophyll cells is between 20 and 30 ,u, with spinachmesophyll cells being about 30 to 45 4u in diameter. One reason

the yield is low may be because of the large vacuole and lesschloroplast per unit volume when compared to crabgrass or

spinach as seen in Table IV. S. telephium leaves also are very

fleshy and crush easily when pressure is applied, so presumablythe cells break easily. Table IV also summarizes some generalcharacteristics of CAM leaves, compared with other plants,

M I N U T E S

FIG. 4. CO2 fixation by S. telephium spongy mesophyll cells dur-ing light (Q) and dark (0). The dotted line shows endogenous ac-

tivity and the solid lines denote the presence of PEP (10 mM).

12

10

cr

0 250 Soo 730 *x0CHLOROPHYLL IN REACTION MIXTURE (I9A"9I)

:5

00

1e

25. ~ ~~ ~ ~ ~ ~ ~ ~ ~ 1~

FIG. 5. Effect of chlorophyll concentration of CO2 fixation inthe presence of PEP (10 mM) with isolated spongy mesophyll cellsfrom S. telephium leaves. Reactions were incubated at 2500 ft-c andat 30 C.

Plant Physiol. Vol. 51, 1973too

i5WU



Plant Physiol. Vol. 51, 1973

demonstrating their succulent nature and their low chloro-phyll content.

There are some methods available for decreasing the amountof acidity in CAM plants. By increasing temperature in thenight, the acid accumulation at night is diminished (7, 18).Changes in the duration of light, such as exposure of leaves

7 SHa

FIG. 6. Effect of pH on CO2 fixation with isolated spongy meso-phyll cells from S. telephium leaves in light (2500 ft-c) at 30 C, with10 mM PEP, 50 mM Tricine NaOH buffer (M) and 50 mM HEPESNaOH buffer (A). The pH values indicate the pH of the suspendingmedia.

140

w I"

VIw8 20

4 20

200

2:70

5C6

_

a sly4C

0

to

620

10 20D

for at least 24 hr to light (18) will lower the acid content. An-other method that has been used is to infiltrate leaves with 1%ammonium hydroxide solution under vacuum, which shouldpenetrate the leaves and decrease the acid level. In this study,the acid content was reduced by using leaves which had beenin light for 6 to 8 hr prior to starting cell isolation. In ourstudies with whole leaves, the titratable acidity at night wasabout 16 meq/ 100 g fresh weight, while in the light it droppedto 5 to 7 (20).From the first experiments with the isolated cells from S.

telephium, it was clear that CO2 assimilation was occurringat lower rates than with intact illuminated leaves. Intactleaves at 2500 ft-c assimilated CO2 at rates ranging from 70to 85 1imoles of C02/mg chl-hr (20). The endogenous ratesof CO2 fixation with the isolated cells varied from about 3to 14 umoles of CO2 fixed/mg chl-hr in day-to-day experi-ments (20). Other experiments with various exogenous sub-strates, however, demonstrate that the isolated cells can as-similate CO2 with good activity for CAM tissue. Light versusdark experiments with isolated cells usually indicated a smallstimulation in the light (Table III). Experiments are in prog-ress on identifying the product of "4C02 fixation in light anddark. We conclude from the present experiments that the cellswere as active as the intact leaf in fixing CO2 on a chlorophyllbasis.

30

TEMPERATURE t40 50 00

B

Ea= 11-48 K. CAL/MOLE \

3-0 3.1 3.2 3.3 _3.4 331 -3T x 10

FIG. 7. Effect of temperature on CO2 fixation with spongy mesophyll cells from S. telephlium leaves (A). Arrhenius plot of temperature effecton CO2 fixation (B). Studies in light (2500 ft-c) with 10 mm PEP. The temperatures are those of the bathing medium around each reaction mix-ture.

A

I.

SEDUM LEAF CELLS 101




154

crv

o 64

PEP COCENTRATIO, M X W

FIG. 8. Dependence of CO2 fixation upon PEP concentration inspongy mesophyll cells of S. telephium leaves during fixation inlight (2500 ft-c) at 30 C.

RWOS 5-P CONCENTRATION M X

FIG. 9. Dependence of CO2 fixation upon ribose-5-P and MgCl2concentration with spongy mesophyll cells from S. telephium leavesduring fixation in light (2500 ft-c) at 30 C. 0: 10 mM MgCl2; 0: 5

mM MgCl2; +: 0.1 mM MgCl2.

Classically cells and membranes surrounding cells arethought of as being fairly impermeable to many intermedi-ates of carbon metabolism, particularly phosphorylated in-termediates. Thus, it has been a surprising finding with iso-lated leaf cells to learn that many intermediates have effectson CO2 fixation when added to the medium bathing isolatedleaf cells (10, 20, 21).

S. telephium leaf cells respond quite markedly to the ad-dition of exogenous substrates with PEP having a very pro-

nounced stimulation upon CO2 fixation (Table III and Fig.8). The stimulation of CO2 fixation upon addition of PEP tothe media may be interpreted as confirming the presence ofPEP carboxylase in these cells. Apparently, light had littleeffect on this activity. Using this activity, it was establishedthat these cells behave in a standard fashion in that the reac-

tion is: (a) linear with time (Fig. 4); (b) linear with cell (enzyme)concentration (Fig. 5); (c) a definite pH response was noted(Fig. 6); (d) a temperature response curve similar to some

enzyme catalyzed reactions was obtained (Fig. 7); (e) chang-

ing substrate concentration results in a standard enzyme versussubstrate concentration curve (Fig. 8); (J) the activity wassensitive to boiling the cells for 5 min (Table III and experi-ment II).By adding 2-PGA, the precursor of PEP to the cells, one

also can assume that enolase is quite active in the cells (Ta-ble III and ref. 20). However, when 3-PGA was added to thecells (20), the rate of CO2 fixation was only one-tenth theactivity when feeding PEP, which could be interpreted as indi-cating that 3-PGA mutase has a low activity or has been in-activated in the isolation of cells, and alternatively 3-PGAdoes not readily penetrate the cells. The present data do notwarrant a choice between these possibilities, but from otherexperiments, penetration seems to be the problem (unpub-lished data).When RuDP was used, the fixation of CO2 was substan-

tially stimulated to values of about 60 ,umoles C02/mg chl hr,and the rate did not seem to be affected by illumination (TableIII). Also compounds such as R-5-P and 6-PGA, which areclosely involved with the oxidative pentose cycle, were ef-fective in promoting CO2 fixation in suitable reaction mix-tures (Table III). Attaining a proper Mg2` to R-5-P ratio(5:1, Fig. 9) has been noted previously (1, 13). The simplestinterpretation of these results is that RuDP carboxylase is ac-

tive in these isolated cells as is R-5-P isomerase and Ru-S-Pkinase. Thus one could postulate that a portion of the pentosecycle was operative.The experiments on cellular CO2 fixation were designed

to disprove or to collect supporting data for the scheme givenin Figure 3. With the information collected, the light anddark aspects of CAM CO2 fixation can be fitted into per-spective. Figure 3 summarizes the pathways for net CO2 fixa-tion in CAM plants in light and in dark. Section A, whichcontains the reactions to the left of the solid line drawnthrough the figure, occurs both in the dark and in the light.Section B occurs primarily in the light, particularly the pho-tosynthetic reductive pentose phosphate cycle and the forma-tion of PEP from pyruvate. In the light, the entire schemein Figure 3 may operate, and in the dark only section A is

Table IV. Comparisont of Area, Percentage of Dry Weight,Thickness, anid Chorophyll Contelit of Leaves from CAM,

C4 Cycle, and Penttose Cycle PlanitsThese values are the average of three determinations.

CAM'CactusCrassula argeniteaSedum telephoidesSedum telephiumBryophyllum cre-

niata

Bryophyllum dai-gremontiana

C4 cycle Crabgrass2Pentose cycle Spin-

ach3

AverageLeafArea

dm2/g

fresh wt

0.01730.05240.0628

i0.0882

0.0608

0.4438

Leaf

Drywt

2.866.473.555.406.74

5.08

22.39.8

Chl/gFreshwt

I mg

0.2380.4120.4300.4600.300

0.888

Typical Leaf Thickness

Tip Middle Base

MfM,fl

2.701.000.800.75

3.101.100.820.92

1.30 1.68

2.320 0.18 0.181.420 0.82 0.82

3.501.200.951.40

1.90

0.180.82

I Plants grown in greenhouse at about 5000 ft-c.2 Field-grown plants, midsummer in Athens, Ga.3 Purchased from local market.

e ( tKm :0.7 x13M /

3- ~~~~~~-3

1.0 0.66 0433 0 0433 06 1o 1.33 16

T

0a 12 -16 20 2 28 32 36 40

102 Plant Physiol. Vol. 51, 1973



operative. Clearly there are many reactions and enzymes insection A and section B which are identical or which aresimply reversed. Thus, the direction(s) of flow most certainlyare highly regulated, but this problem has yet to be investi-gated in sufficient detail for any definite statements to bemade, although some workers have proposed that such en-vironmental factors as temperature are definite regulators(7). We have experiments in progress on identification of theproducts of '4CO2 fixation which show that the predicted la-beled products of both light and dark metabolism in Figure 3can be observed with these isolated S. telephium cells.

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Plant Physiol. Vol. 51, 1973 SEDUM LEAF CELLS



isolation mesophyll cells from telephium · pdf filemncl2, 2 mmnano.,, 5 mmmgcl2. 5 mm ... the...

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