Black, Clanton C.

Kenyon, William H.

Kringstad, Randi

Elsevier - Publisher Connector 

Diurnal changes in the malic acid content of vacuoles isolated from leaves of the crassulacean acid metabolism plant, Sedum telephium 

Volume 94, number 2 FEBS LETTERS October 1978 
DIURNAL CHANGES IN THE MALIC ACID CONTENT OF VACUOLES ISOLATED FROM 
LEAVES OF THE CRASSULACEAN ACID METABOLISM PLANT, SEDUM TELEPHIUM 
William H. KENYON, Randi KRINGSTAD* and Clanton C. BLACK 
Department of Biochekry, University of Georgia, Athens, GA 30602, USA 
Received 31 July 1978 
1. Introduction 
The accumulation of malic acid at night in green 
cells followed by malic acid depletion the next day is 
characteristic of Crassulacean acid metabolism (CAM) 
plants. The diurnal changes in malic acid content are 
accompanied by reciprocal changes in starch content 
with starch providing the carbon skeleton for phos- 
phoenolpyruvate, the CO* acceptor at night. Malic 
acid is decarboxylated during the day to provide COZ 
for photosynthesis [ 11. In contrast, other organic 
acids such as isocitric do not exhibit a daily fluctua- 
tion in levels [2]. With the realization that massive 
amounts of malic acid accumulate at night [ 1,2], it 
was widely postulated that malic acid is stored in the 
large vacuoles of green CAM cells. Indeed, calculations 
based on Bryophyllum leaf cell volumes and the 
amount of acid accumulated at night showed that ‘if 
malic acid were confined to the cytoplasm, its concen- 
tration would be 7-8 M, but if it were in the vacuole 
also, its average concentration would be 0.25 M’ [3]. 
Thus, to prevent a variety of deleterious effects of 
acidic conditions on cytoplasmic components, a green 
CAM cell would in theory temporarily sequester 
malic acid in its vacuole at night. However, data to 
test this hypothesis are not available. 
Recently, techniques have been developed for 
isolating large quantities of vacuoles from plant tissues 
[4-71. Isolated Bvophyllum leaf vacuoles [6] con- 
tained 0.002-0.02 nmol malic acid/vacuole which 
comprised the bulk of the malic acid present. However 
* Permanent address: Institute of Pharmacy, University of 
Oslo, Oslo 3, Norway 
Elsevier/North-Holland Biomedical Press 
the daily malic acid content of vacuoles was not inves- 
tigated. Using the CAM plant Sedum telephium, we 
have isolated intact vacuoles from leaf protoplasts 
with yields -20%. In the present investigation we have 
used these isolated vacuoles to determine the cellular 
localization of malic and isocitric acids and to study 
the diurnal pattern of malic acid accumulation and 
depletion. These diurnal changes in malic acid are 
interpreted as showing that the vacuole is directly 
involved in the CO2 assimilation pathway of green 
CAM plants. 
2. Materials and methods 
Sedum telephium L. was cultivated in growth 
chambers under 15 h day at 30°C and 9 h night at 
15°C with an irradiation intensity of 150 (uE rnm2.se1 
at the upper left surface. Plants were well watered 
and fertilized under conditions which support active 
CAM [8]. Leaves were harvested throughout a day at 
the times indicated and protoplasts were isolated by 
digestion with commercial preparations of pectinase 
and cellulysin. Pure vacuoles were isolated within 2 h 
on Ficoll gradients using modifications of procedures 
in [7]. Protoplasts and vacuoles were counted and 
checked for purity microscopically. Further details of 
the isolation and purification of vacuoles and proto- 
plasts will be presented separately (in preparation). 
Leaf titratable acidity was measured as in [8]. Malic 
acid was assayed using chicken liver malic enzyme 
purchased from Sigma Chem. Co. [9]. Isocitric acid 
was assayed with porcine heart isocitric dehydrogenase 
purchased from Calbiochem. Corp. [lo]. The iso- 
281 
Volume 94, number 2 E’EBS LETTERS October 1978 
citric lactone ring was opened prior to assay by adjust- 
ing the extracts to pH 12 and heating at 60-70°C for 
30 min. The extracts were readjusted to pH -10 and 
immediately assayed. 14C02 labeling experiments 
were performed by sealing leaves in Mylar bags, inject- 
ing 75 j.&i 14C02 into the bags, and allowing the leaves 
to fix the r4C02 for 30 min. Dark r4C02 labeling was 
done at 21°C in complete darkness while light label- 
ing was at 3 1°C and at 440 /.IE mm2.s-r. Vacuoles 
were isolated from these leaves immediately following 
these 14C02 incubation periods. These labeling times 
were selected so that acid was accumulating at night 
or the acid was at the lowest daily level [8]. 
3. Results and discussion 
An experiment illustrating the data obtained on 
malic acid content of isolated vacuoles at timed inter- 
vals throughout a day is presented in fig.1. The malic 
acid content of isolated vacuoles reaches a low value 
each light period -0.02-0.04 nmol malic acid/vacuole. 
Subsequently the vacuole malic acid content rises to 
peak values -0.5-0.75 nmol/vacuole at the end of 
the night period. In the protoplasts used to isolate the 
MALIC ACID 
ISOLATED 
VACUOLES 
ISOLATED -1 + 
I I I I I I I I 
15 18 21 24 3 6 9 12 
HOUR OF DAY 
Fig.1. The diurnal pattern of leaf titratable acidity (0); and Fig.2. Diurnal changes in the molar ratio of malic acid to 
the malic acid content of isolated leaf vacuoles (0) and iso- isocitric acidsin isolated vacuoles (0); isolated protoplasts (0); 
lated leaf protoplasts (a) of Sedum felephium. and intact leaves (a) of Se&m ielephium. 
vacuoles, the malic acid content was similar to the 
vacuole content except at the end of the night (fig. 1). 
There we had an experimental problem of partial 
breakage of protoplasts and vacuoles, presumably due 
to their high acid content, so that accurate counts 
were difficult. Our vacuole isolation procedures were 
developed in low acid and we have not yet investigated 
the breakage problem when the acid content is high. 
Data are given in fig.1 on leaf titratable acidity show- 
ing that these Sedum leaves were in CAM [8]. All of 
these experiments have been repeated several times 
without discrepancies arising in the daily patterns. 
Since CAM leaves are known to have high contents 
of isocitric acid [ 111, we assayed its daily pattern in 
intact leaves, in isolated protoplasts, and in isolated 
vacuoles. In contrast to malic acid, the vacuole content 
of isocitric acid showed little change throughout a day 
and no consistent pattern of change relative to night 
or day. The isocitric acid content of vacuoles was 
0.12 f 0.07 nmol/vacuole. Isocitric acid also did not 
show a pattern of diurnal change in leaves or in proto- 
plasts. These studies confirm earlier work consistently 
showing that isocitric acid does not fluctuate in intact 
CAM leaves [ 11,121 and show that isocitric acid is also 
stored in the vacuoles of green CAM cells along with 
malic acid. 
Since isocitric acid does not fluctuate in a diurnal 
fashion in leaves or isolated vacuoles we reasoned it 
could provide an internal standard on which to base 
ISOLATED I 
HOUR OF DAY 
Volume 94, number 2 I:EBS LETTERS October 1978 
Table 1 
Accumulation pattern of 14C in cellular components isolated 
from Sedum telephium leaves fed 14C0, in the dark or light 
Time of leaf Recovery of 14C from a vacuole purification 
“C0, fixation ~ 
Purified Insolublesa SolubIesb 
vacuoles 
(% total ‘Y in protoplasts) 
- 
04.00-04.30 49 5 46 
in dark 
16.00-16.30 - 4 92 -4 
in light 
a This purification fraction contains chloroplasts, starch and 
other insoluble cellular components. Over 99% of the 
chlorophyll is in this fraction 
b This is a compilation of all purification fractions except the 
vacuoles or insolubles and is comprised of the soluble 
materials derived from broken protoplasts, vacuoles and 
other organelles 
the malic acid content of vacuoles. We calculated the 
ratio of malic to isocitric acid, and fig.2 gives data for 
isolated vacuoles, isolated protoplasts, and intact 
Sedum leaves. The results show a similar diurnal 
pattern of malic acid accumulation and depletion 
among all three (fig.2). We interpret the data in fig.l,2 
as showing that malic acid fluctuates in a diurnal 
fashion in CAM leaves and that the vacuole is its 
major site of accumulation at night. 
In support of this hypothesis we also fed 14C02 to 
Sedum leaves in the light or in the dark and then 
immediately isolated the vacuoles. The Ficoll gradi- 
ents were fractionated and individual fractions were 
assayed for both 14C and malic acid. As shown in 
table 1, 14C accumulated in the vacuoles at night 
while less than 5% of the 14C was in the vacuole during 
the day. This vacuole 14C was shown to be in malic 
acid. Little 14C accumulated in insoluble materials 
during the night but 92% of the 14C accumulated in 
this fraction during the day (table 1) showing the 
predominance of photosynthetic CO* fiiation rather 
than fi-carboxylation during the day. 
4. Conclusion 
At night CAM tissues primarily fs CO2 via the 
/3-carboxylation of PEP and subsequently store the 
malic acid in the vacuole of green cells. Malic acid is 
depleted during the next day via removal from the 
vacuole followed by a cytoplasmic decarboxylation 
of malic acid in Sedum, or of oxaloacetic acid in 
other CAM plants [ 131, with the CO? being used in 
photosynthesis. Isocitric acid also is stored in CAM 
leaf vacuoles but it does not show a constant pattern 
of daily change in concentration. These studies sub- 
stantiate the long standing hypothesis [ 1,3] that 
malic acid in CAM plants is temporally stored in the 
vacuole and they clearly implicate the vacuole as a 
cellular organelle with an active role in the net fixa- 
tion of COz in CAM plants. 
Acknowledgements 
This research was supported by NSF grant PCM 
7708548. W. H. Kenyon is the recipient of a Graduate 
Assistantship in Botany and R. Kringstad is the 
recipient of travel grants from Norges Almenvitens- 
kapelige Forskningsraad, Norsk Farmaceutisk Selskap, 
and the University of Oslo. 
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Diurnal changes in the malic acid content of vacuoles isolated from leaves of the crassulacean acid metabolism plant, Sedum telephium

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