(WITH NINE FIGURES) Received June 28, Bryophyllumof the 11 samples was 7.75 ±0.18 grams per kilo of...

+STUDIES IN THE METABOLISM OF CRASSULACEAN PLANTS:THE EFFECT OF TEMPERATURE UPON THE CULTUREOF EXCISED LEAVES OF BRYOPHYILLUM CALYCINUM

GEORGE W. PUC HER, CHARLES S. L EAVENWORTH, WANDA D.GINTER, AND HUBERT BRADFORD VICKERY

(WITH NINE FIGURES)

Received June 28, 1947

When Bryophyllum calycimum leaves are detached from the plant andcultured in water in darkness at ordinary room temperature, the course ofthe changes in composition with respect to the organic acids, the proteinand the starch depends upon the time of day at which the samples werecollected (5). If leaves are taken in the afternoon at a time of low acidityand low protein content, there is a prompt increase both in acids and inprotein and a corresponding decrease in the high starch content. Alterna-tively, if the leaves are collected in the morning at a time of high acidity andprotein content, both acids and protein fall and the starch increases. Thesechanges take place within the first 24 hours and appear to be expressionsof the normal metabolism of this species. After 24 hours, certain otherchanges begin to make their appearance; in particular, for leaves collectedin the afternoon, the acids and the protein, after reaching maxima, begin tofall and the starch may increase during the second day of the culture period.It seems possible that this reversal in the direction of the chemical changesrepresents the initiation of the catabolic changes that inevitably follow ex-cision of a leaf. Accordingly, in order to study this possibility further, anexperiment was carried out in which the culture period at 200 C. was pro-longed to 6 days and, in addition, parallel cultures were carried out at90 C. and at 10 C. in the hope that, at the lower temperatures, the reac-tions might proceed more slowly and certain of the details might be revealedmore clearly. The present paper gives a report of this experiment.

Methods

SEIECTION OF SAMPLES

Bryophyllum calycirwm plants were potted in sand on October 7, 1942and were grown in the greenkouse upon a nitrate-containing culture solu-tion (See (4) for details of composition). In the afternoon of June 2, 1943,a hot sunny day, 30 plants were selected and about 500 young leaflets werepicked from the second to the fourth leaf, counting from the top of theplants. The leaflets were taken to the laboratory and eleven samples of 40leaflets each were rapidly chosen at random and were then adjusted, by theinterchange among the samples of at most 5 leaflets, so as to be of equalweight. The mean freslj weight of the samples was 134.3 + 0.2 grams.

123

www.plantphysiol.orgon February 23, 2020 - Published by Downloaded from Copyright © 1948 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

PLANT PHYSIOLOGY

Two samples were at once placed in the dryino oven at 800 C. and subse-quently prepared for analysis (4), while the others were arranged in woodenracks with the bases of the leaflets immersed in water in enamel-ware trays,three samples being placed in each of three completely dark rooms the tem-perature of which was controlled, respectively, at 200, 90, and 10 C. Allsamples were in position by 4 P.M. (standard time) which was taken as thezero time of the experiment.

Fundamental data on the individual samples are shown in table I whichalso gives information on the times that samples were withdrawn for analy-sis. The analytical data were computed by means of factors derived from

TABLE IFUNDAMENTAL DATA ON SAMPLES OF EXCISED YOUNG Bryophyllum calycinum LEAFLETS

SUBJECTED TO CULTURE IN WATER IN DARKNESS AT THREE DIFFERENT TEM-PERATURES. FIGURES NOT OTHERWISE DESIGNATED ARE GRAMS.

EACH SAMPLE CONSISTED OF 40 LEAFLETS COLLECTEDIN THE AFTERNOON

FRESH FRESH CRUDE CRUDE DRY FRESH WEIGHTHOURS WEIGHT: WEIGHT: DRY WEIGHT PER KILO:CULTURED START END WEIGHT PER KILO END

CONTROL SAMPLES

0 134.3 13.0 96.80 134.5 12.7 94.4

CULTURED AT 200 C.

23 134.0 143.5 12.9 96.3 107166 134.2 142.1 12.6 93.9 1059

142 134.2 131.0 11.9 88.7 976

CULTURED AT 90 C.

23 134.2 144.9 13.4 99.8 108066 134.6 149.0 13.3 98.8 1107

142 134.5 148.9 12.5 92.9 1107

CULTURED AT 10 C.

23 134.2 142.2 13.0 96.9 106066 134.6 141.2 13.2 98.1 1049

143 134.1 122.5 12.7 94.7 914

these figures so as to show the quantities of each component that would havebeen found if each sample had weighed exactly 1 kilo at the start of theexperiment.

The set of samples cultured at 200 C. was designed to furnish a controlat approximately normal room temperature and this test was essentially aduplicate of the previous experiment carried out with leaves picked in theafternoon (5). The chief difference is that the leaflets of the present ex-periment were selected from the youngest fully developed leaves on theplants, whereas, in the earlier experiment, the samples represented all of thecompound leaves, that is, all leaves exclusive of the 6 to 9 simple leaves thatoccupy the lowest position on the stem in thisspecies.

124



PUCHER ET AL.: MiETABOLISM OF BRYOPHYLLUM 125

The limits within which the samples initially duplicated each other incomposition can best be appreciated from the data for ash and total nitrogenplotted in figures 1, 2, and 3. It would not be expected that either of thesecomponents would be altered by the treatment. The mean value of the ashof the 11 samples was 7.75 ± 0.18 grams per kilo of original fresh weight,the standard deviation being + 2.3% of the mean. The mean value fortotal nitrogen was 2.47 + 0.10 grams per kilo, the deviation being + 4.1%oof the mean. Accordingly, in spite of the fact that the original fresh weightof the samples varied less than 0.2% as a result of the method of selection,the coefficients of variation with respect to nitrogen and ash were preciselythe same as those observed in the previous experiment (5) where no attemptwas made to equalize the initial fresh weights of the samples. It may there-

Fln. 1 Fig. 2 Fig. 3

Cultured at 20@ C. Cultured at 9- C. Cultured at l C.

Darkness Darkness Darkness

Total NTotal N Total N

Prot~ein ~NProtei

10 010 10

5ibAsh 5 Ash

Sr. 40 80 120 160 Ers. 40 80 1*0 160 ru. 40 80 180 160

FIGS. 1 to 3. Nitrogen, protein, and ash content of excised leaflets of Bryophyllumcalycinum cultured in water in darkness respectively at 200, 90, and 10 C. Data are

expressed in grams per 1 kilo of original fresh weight.

fore be assumed, as before, that differences in composition substantially inexcess of ± 4% observed for other components are results of the treatmentto which the leaves were exposed.

Results

PROTEIN

The effect upon the protein of the temperature at which the samples werecultured in darkness is shown in figures 1, 2, and 3. No change was detectedat 200 C. during 66 hours but the sample analyzed at 142 hours showed adefinite decrease. Unfortunately, no sample was analyzed at the 14-hourpoint so that the phenomenon of increase in the protein shortly after theleaves were placed in darkness (5) was not observed. However, there wasa slight increase at 23 hours in the samples cultured at 90 C. and a rise inthe curve is also apparent at the 66-hour point in the sample at 1° C. Theseincreases are respectively 6% and 5% of the initial value and are thus at



PLANT PHYSIOLOGY

the limit of significance; nevertheless, if it be assumed that chemical reac-tions within the cells are slowed down by the decrease in temperature, thebehavior observed is consistent with what would have been anticipated fromthe previous experiment.

The decrease in protein at 142 hours in the experiment at 200 C. is like-wise to be expected from analogy with the behavior of the protein of tobaccoand rhubarb leaves (7, 8). During prolonged culture of detached leaveseither in darkness or in light, decomposition of the protein owing to hydroly-sis by proteolytic enzymes is invariably encountered. With tobacco, thiscan be demonstrated to have been initiated within 24 hours but the proteinof Bryophyllum leaves is more slowly attacked; definite evidence of prote-olysis was not secured at the end of 48 hours in the previous experiments (5).The present case is clear, however; the decrease in protein nitrogen at 142hours was almost 18% of the initial value. The decrease at 142 hours in theexperiment at 90 C. was nearly 8% and thus also suggests that proteolyticaction had begun to become significant at this point. At 10 C., however,there was no indication of the initiation of proteolysis even after 143 hours.

ORGANIC SOLIDS

The lower curves in figures 4, 5, and 6 show the behavior of the organicsolids at the three temperatures. No change was observed during the firstday of the culture period, but statistical examination of the results' at 23,66, and 142 hours for the three tests showed that there is less than one chance

Fig. 4 Fig. 5 Fig. 6Cultured at 200 C. Cultured at 90 C. Cultured at le C.


6 6.6

5 PR 5, ~~~~~~~~~~~~~~~~~pH

4 4 4

3 3 3

80 Organic Solids 8080

Organic SolidsOrganic Solids

70 70 70Hrs. 40 80 120 160 Ers. 40 80 120 160 Irs. 40 80 120 180

FIGS. 4 to 6. pH, organic solids, and organic acids of excised leaflets of Bryophyl-lum calycinum cultured in water in darkness respectively at 200, 90, and 10 0. Data areexpressed in grams per 1 kilo of original fresh weight.

in 50 that the organic solids are not a diminishing function related to time.Thus, loss of organic solids by uncompensated respiration, although slow,does occur during prolonged culture of Bryophyllum leaves in darkness.Moreover, the rate of loss at 90 C. was scarcely perceptibly less than thatat 200 C. although that at 10 C. was apparently slower.

1 We are indebted to DR. C. I. BLISS for this analysis.

126



PUCHER ET AL.: METABOLISM OF BRYOPHYLLUM

ORGANIC ACIDS AND CARBOHYDRATES

The upper curves in figures 4, 5, and 6 show the effect of culture on thepH of the leaves. At 200 C., there was a drop of 1.4 units during the firstday owing to the prompt synthesis of organic acids in the leaves when firstplaced in darkness and, at 90 C., the drop was 1.7 units in the same period.At 10 C., the drop was only 1.1 units. The subsequent course of the curveswas different in each case. At 200 C., the pH slowly rose, suggesting thatorganic acids were slowly decomposed as the culture period progressed; thisbehavior is similar to that previously observed (5). At 90 C., however, thepH remained at about the same low level throughout the entire period afterthe first day of the experiment, suggesting that decomposition of organic

Fig. 7 Fig. 8 Fig. 9Cultured at 201 C. Cultured at 9g C. Cultured a; 10 C.


30 ' 30 30

ToalOganic Acids

Ttl25 58 25 - Organic Acids

Total OrganicAcids

20 80 20 -

15 15 15 Isocitric

10 Ir Isocitric 10 108 14 ic

I

Citric Caferb.\Solab~-exprsse5 81Crmp

~~~~~ C~~~~~a

I A~~~~Era. 40 80 120 160 Hra. 40 80 120 160 Era. 40 80 120 1*0

FIGs. 7 to 9. pH, organic acids, and carbohydrates of excised leaflets of Bryophyl-lum calycinum cultured in water in darkness respectively at 200, 90, and 10 C. Data areexpressed in grams per 1 kilo of original fresh weight.

acids did not occur; while at 10 C., the pH continued to decrease for 66 hoursand then remained essentially constant.

The curves for total organic acids in figures 7, 8 and 9 correspond fairlyclosely with the inferences to be drawn from the curves for pH. The markedincreases during the first day at all three temperatures are evident, but themost striking feature is that the increase was larger at the lower tempera-tures than at 200 C.; it would appear that the position of equilibrium wasdisplaced in the direction of a higher concentration of organic acids at lowtemperatures.

127



PLANT PHYSIOLOGY

The behavior of the starch (plotted as a broken line in the figures) was,in each case, roughly symmetrical with that of the total organic acids. At200 C., starch decreased during the first day by 8.4 grams while 11 gramsof organic acids were being formed. During the remainder of the cultureperiod, organic acids diminished by 5.1 grams while starch increased by 0.5gram. This increase was too small to be significant, but the phenomenonresembles that previously observed (5) where an appreciable quantity ofstarch appeared during the second day of a similar culture experiment at atime when the organic acids were diminishing.

At 90 C., 17 grams of organic acids were synthesized in the first daywhile 11.1 grams of starch disappeared; subsequently there was no changein either total acids or starch. At 10 C., the symmetry of the curves foracids and starch is especially well marked for the first 66 hours; during thisperiod, 16 grams of acids were formed while about 6 grams of starch dis-appeared. During the last 3 days of the culture period, however, the totalorganic acids dropped by about 2 grams and starch also dropped by 2.4grams.

Perhaps the most important inference from these figures is that in allcases the quantity of starch consumed during the first day was less than thequantity of organic acids produced. Although there seems little doubt thatthe starch contributed largely to the formation of the acids, and at 9° C.was ultimately almost exhausted, other components of the tissue were almostcertainly drawn into the reactions. Nevertheless, the change in the solublecarbohydrates (plotted in the figures as a broken line connecting filledcircles) was too small to account for the difference. The experiment at 200C. differs in this respect from the previous culture experiment (5) in whichthe loss of starch from a sample of leaves cultured under similar conditionsconsiderably exceeded the increase in organic acids. In that case, it waspossible to conclude that the organic acids may have been derived entirelyfrom the metabolism of the starch; in the present case, a similar conclusioncannot be drawn. Thus, the relationship between the acids and the starchis by no means a simple one and the calculation of stoichiometric quantitiesis obviously futile; starch may be the source of much of the acid, in certaincases of all, but it is not the only possible source.

The details of the changes in the organic acid composition of the samplesare also plotted in figures 7, 8, and 9. At 20° C., there was a prompt andextensive synthesis of malic acid during the first day followed by a rapiddecline. Meanwhile, citric acid accumulated in the tissues during 66 hoursand then remained at a constant level for the rest of the experimental period.Isocitric acid was high at the start and underwent a rather small drop fol-lowed by a rise. The relationships among these acids after 23 hours is in-dicative of transformation of malic into citric and isocitric acids coupledwith disappearance of a small part of the acidity.

At 90 C., malic acid production at the expense of starch was stimulatedduring the first day in comparison with the behavior at 200 C.; subsequently,

128




malic acid appears to have been extensively transformed into citric acidalthough to only a small extent into isocitric acid. There was only a negli-gible loss of total acid. In both experiments, starch decomposition ceasedafter the first day but, at 90 C., nearly all of the starch had been consumedand there was a detectable increase in soluble carbohydrates in the intervalbetween 23 and 66 hours. Only part of this could have arisen from thestarch as the quantity was too great. However, other sources of solublecarbohydrates were doubtless present.

At 10 C., the behavior of the acids was quite different; the increase inmalic acid continued for 66 hours and there was also an increase in isocitricacid, especially during the first day. This is in contrast to the behavior ofisocitric acid in the samples at the higher temperatures; in both of these,isocitric acid decreased during the first day. The most striking point, how-ever, is that malic acid did not decrease during the last days of the cultureperiod; the decomposition of this substance was apparently suppressed at thelow temperature. Instead, isocitric acid decreased while the formation ofcitric acid continued from the start although at a diminishing rate. Theevidence for the transformation of isocitric acid to citric acid is thus espe-cially clear. The drop in total acidity during the last 4 days appears to havearisen from the decomposition of isocitric acid and was only in part com-pensated for by the increase in citric acid.

Starch continued to decrease throughout the experiment, finally almostcompletely disappearing and, whatever its fate may have been after 66 hours,it was not converted into organic acids, or, if so, the reactions continuedbeyond the organic acid stage. As at 90 C., there was a small but probablysignificant increase in soluble carbohydrate in the interval between 23 and66 hours, followed by a small drop.

Discussion

Perhaps the most striking feature of these experiments is the lack ofevidence of a temperature coefficient of the anticipated order of magnitudefor the reactions that occur during culture. Ordinarily, one expects a de-crease of 100 C. to result in a reaction rate of about one-half of that at thehigher temperature. On the contrary, in the present experiments, a dropof 110 C. substantially increased the quantity of total organic acids pro-duced in 23 hours, increased the quantity of malic acid formed in the sameperiod, and increased the quantity of starch consumed. The quantity ofcitric acid formed in the first day was indeed almost exactly twice as greatin the experiment at 200 C. as at 90 C., but the total quantity formed in 143hours was nearly twice as great at 90 C. as at 200 C. and a further drop intemperature to 10 C. diminished the total quantity of citric acid formed onlyto the level of the experiment at 200 C. In other words, culture of Bryo-phyllum leaves at 90 C. appears to stimulate the formation of citric acid ascompared with higher or lower temperatures. Wolf (10) has already

129



PLANT PHYSIOLOGY

recorded the observations that culture at 7 to 80 C. appears to stimulate totalacid production and (9) starch utilization.

The decrease in temperature from 90 to 10 C. diminished the rate of totalacid production appreciably, 66 hours being required to produce as muchacid at the lower temperature as was produced in 23 hours at the higher.This relation gives a temperature coefficient for the over-all reaction of ap-proximately the anticipated order of magnitude. It is also true that thequantities of malic acid formed and of starch consumed in 23 hours at 90 C.were about twice the quantities at 1° C. and that the total quantity of citricacid formed in 142 hours at 90 C. was only slightly less than twice thatformed in about the same time at 10 C. But here the easily perceived regu-larities cease; Wolf's statement that the positions of the reaction equilibriaare "streng temperaturabhhngig" is undoubtedly correct and it seems clearthat to attempt a more detailed analysis of the situation in the absence offuller data as well as of information concerning the enzyme systems would,at the present time, be premature.

There is no doubt that many different equilibria are concerned. Onlya few of the individual reactions stand out clearly as, for example, the con-version of malic to citric acid shown by the curves of figure 8. This isa reaction already well known to take place in tobacco leaves (6, 7) undersomewhat similar conditions. The evidence that starch is a source of theorganic acids synthesized in darkness also seems unequivocal. But the posi-tion in the general scheme of reactions of isocitric acid, for example, is espe-cially puzzling and there is no hint whatever in the present data of anexplanation of why malic acid should play the dominant role in the trans-formations that occur at the start of the culture period.

That the acid metabolism of crassulacean plants is affected by the tem-perature at which the studies are made has long been known. The earliestexperiments were apparently those of DE VRIES (2) and the field up to 1932has been reviewed by EVANS (3). BENNET-CIARK (1) has pointed out thatthe transformation of carbohydrate to malic acid is a reaction that liberatesenergy and accordingly the production of acid should be promoted by adecrease in temperature. This was found by him to be the case for Sedumpraealtum and has also been noted by Wolf for Bryophyllum. The presentexperiments are in agreement with respect to the temperature range from200 to 90 C. but there is a limitation inasmuch as further decrease in tem-perature does not lead to still greater production of acid as is clear fromfigures 8 and 9. The position of the equilibrium is thus controlled by fac-tors in addition to temperature as the freezing point of water is approached.

Although the explanation of biochemical events in terms of physico-chem-ical relationships is the goal of investigations such as these, only incompletesuccess can be anticipated unless the physical chemical systems themselvesare isolated. It is manifestly insufficient merely to isolate the tissue as hasbeen attempted in the present instance; the reactants themselves and the

130




catalysts of the reactions, the enzymes with their various co-factors, must beisolated before a full explanation of the events will be forthcoming.

Summary

Samples of young leaflets of Bryophyllum calycinum picked in the after-noon were cultured in water in darkness respectively at 200, 90, and 10 C.,and determinations of the organic acids and starch were made at intervalsover a period of 6 days. At 200 C., the total organic acids increased mark-edly for the first day and subsequently decreased slowly; the changes inmalic acid accounted for much of the change but citric acid increased at firstrapidly and then slowly throughout the experimental period; isocitric acidchanged very little. Starch decreased during the first day and then re-mained nearly constant.

At 90 C., total organic acids increased during the first day more rapidlyand to a greater extent than at 200 C., malic acid again being mainly re-sponsible; subsequently the total acids remained constant but malic aciddecreased, its place being taken by citric acid and, to a minor extent, byisocitric acid. Starch nearly disappeared during the first day and remainedat a low level throughout.

At 10 C., the increase in organic acids was somewhat slower than at 90 C.but was almost equally as extensive. All three of the main organic acidsshared in the increase although malic acid played the largest role. Malicand citric acids rose to maxima that were maintained throughout, but iso-citric acid decreased moderately during the last 3 days of the experiment.Starch decreased throughout although more slowly than at 90, and finallyalmost disappeared. In none of the experiments was there any substantialchange in the soluble carbohydrates, nor was the loss of starch quite suffi-cient to account for all of the organic acid formed; thus although starch isdoubtless the main component that contributes to the formation of acids, itis not necessarily the only one.

The increased formation of organic acids at 90 C. as compared with 200C., following BENNET-CLARK, is tentatively ascribed to the fact that theoxidation of carbohydrate to organic acids is a reaction that liberates energyand should accordingly be promoted by decrease in temperature. However,there is a limitation upon this promotion as the freezing point of water isapproached. Because of the complexity of the series of chemical reactionsinvolved, few cases could be perceived in which a decrease of about 100 C. intemperature diminished the extent of the reaction by one-half.

CONNECTICUT AGRICULTURAL EXPERIMENT STATIONNEW HAVEN, CONNECTICUT

LITERATURE CITED

1. BENNET-CLARK, T. A. The role of organic acids in plant metabolism.Part II. New Phytol. 32: 128-161. 1933.

131



PLANT PHYSIOLOGY

2. DE VRIES, H. trber Siiurebildung der Fettpflanze. Bot. Zeitung 42:337. 1884.

3. EvANs, H. The physiology of succulent plants. Biol. Rev. CambridgePhil. Soc. 7: 181-211. 1932.

4. PUCHER, G. W., LEAVENWORTH, C. S., GINTER, W. D., and VIcKiEny,H. B. Studies in the metabolism of crassulacean plants: Changesin the composition of Bryophyllumn calycinum during growth.Plant Physiol. 22: 1-19. 1947.

5. , , , and . Stud-ies in the metabolism of crassulacean plants: The behavior ofexcised leaves of Bryophyllum calycinum during culture in water.Plant Physiol. 22: 477-493. 1947.

6. , WAKEMAN, A. J., and VICKERY, H. B. The metabolismof the organic acids of the tobacco leaf during culture. Jour. Biol.Chem. 119: 523-534. 1937.

7. VICKERY, H. B., PUCHER, G. W., WAKEMAN, A. J., and LEAVENWORTH,C. S. Chemical investigations of the tobacco plant. VI. Chem-ical changes that occur in leaves during culture in light and indarkness. Connecticut Agr. Exp. Sta. Bull. 399. 1937.

8. , , , and . Chem-ical investigations of the rhubarb plant. Connecticut Agr. Exp.Sta. Bull. 424. 1939.

9. WOLF, J. Beitriige zur Kenntnis des Siiurestoffwechsels sukkulenterCrassulaceen. III. Stoffliche Zusammenhiinge zwischen giir-fiihigen Kohlehydraten und organischen Sauren. Planta 28:60-86. 1938.

10. . Beitrage zur Kenntnis des Saiurestoffwechsels sukku-lenter Crassulaceen. IV. Beobachtungen iiber Gehaltsschwank-ungen von Gesamt-, Xpfel- und Zitronensiiure. Planta 29: 314-324. 1939.

132



(WITH NINE FIGURES) Received June 28, Bryophyllumof the 11 samples was 7.75 ±0.18 grams per kilo of...

Documents

Transcript of (WITH NINE FIGURES) Received June 28, Bryophyllumof the 11 samples was 7.75 ±0.18 grams per kilo of...