Temperature and Humidity Effects on Dark C02 Fixation

by Kalanchoe pinnata
ERNESTO MEDINA

Dept. of Biology, Stanford University; on leave from Centro de Ecologia, IVIC, Aptdo. 1827,
Caracas, Venezuela

Received March 16, 1982 . Accepted May 31,1982

Summary
Net CO 2 exchange and acid accumulation during 12 h dark periods were measured in
attached leaves of K. pinnata in a range of leaf temperatures from 12-25 °C and at high and low
leaf-air water vapor deficits. The results showed that
1. Stomatal opening during the dark period was highly sensitive to air humidity at all tempera-
tures tested. Reduction in stomatal conductance resulted in lowered total C02 fixation
integrated over the 12 h dark period;
2. Maximal rates of dark C02 fixation were similar at all temperatures tested within a given
range of leaf-air vapor pressure deficit. However, beginning of net dark CO 2 fixation and
time to reach peak rate of C02 uptake were delayed as temperature increased. Therefore, net
CO 2 uptake and acid accumulated were reduced with increasing temperature during the
dark period;
3. A linear relationship between net C02 fixation and acid accumulation was obtained, its
slope was parallel to the 2 : 1 acid/C02 ratio line.
Key words: Kalanchoe pinnata; acid accumulation; dark COzfixation; temperature and humid·
ity effects.

Introduction
CAM plants frequently show a well defined temperature optimum for total dark
CO 2 fixation, within the 10-20 °C temperature range depending on night tempera-
ture during growth (Wolf 1960; Kluge and Ting 1978; Gulmon and Bloom 1979;
Medina and Osmond 1981). Dark CO 2 fixation clirves at different temperatures pre-
sent a characteristic pattern; near optimal temperature CO 2 fixation begins a few
minutes after onset of the dark period, while at temperatures above the optimum,
there is a marked delay in the initiation of net C02 fixation. The time required to
reach the maximal rate of dark CO 2 fixation is also delayed. Several processes may be
causing this pattern of net C02 fixation, among them, stomata closure (Ting et al.,
1967), impairment of glycolysis (Sutton 1975), consumption of malic acid resulting
from the primary carboxylation reaction (Brandon 1967), or complex effects on the

Abbreviations: CAM: Crassulacean Acid Metabolism; VPD1-a; vapor pressure difference

between leaf and surrounding air.

Z. Pjlanzenphysiol. Bd. 107. S. 251-258. 1982.

252 ERNESTO MEDINA

permeability of tonoplast resulting in leaking of acid accumulated in the vacuole

(Liittge et al. 1975).
Under natural conditions, increasing temperatures are often accompanied by
higher leaf-air vapor pressure deficits, and there are several reports indicating that sto-
mata of CAM plants are very sensitive to this gradient (Conde and Kramer 1975;
Lange and Medina 1979; Osmond et al. 1979).
This paper reports a detailed analysis of temperature and humidity effects on the
gas exchange characteristics and acid accumulation during the night in Kalanchoe pin-
nata. The possible relationships between CAM activity and respiration are discussed
to explain the observed patterns of nocturnal CO 2 fixation.

Material and Methods

Plandets of Kalanchoe pinnata were obtained from leaf buds and cultivated for 3 months in a
green house at Stanford during the spring. Afterwards the plants were transfered to a growth
cabinet with 25/15 0 day-night temperature and 12 h photoperiod; irradiance (400-700 nm) at
plant level was 350 /Lmol photons m- 2 S-I. There was no control of relative humidity in the
cabinet, but the relative humidity never decreased below 60 % during the light period and was
around 80 % during the dark period. Gas exchange measurements began after plants had been
kept for 1 month in the cabinet, and had produced 6-7 leaf pairs. Leaves used for measurements
belonged to the 4-5 th leaf pair. The gas exchange measurements were conducted with an open
system using an infrared gas analyser and a ventilated leaf chamber as described in detail by
Winner and Mooney (1980). The units for CO 2 exchange and leaf conductance used in this
paper are those proposed by Cowan (1977).
C02 and water vapor exchange were measured in attached leaves at several leaf temperatures
ranging from 12-25 oC, and at high or low ambient humidity. At the beginning of the experi-
ment the leaf opposite to the one being measured was cut off and frozen. At the end of the gas
exchange measurement the second leaf was also severed and both leaves were processed for acid
content measurement. Leaves were extracted in boiling ethanol and extracts were titrated with
NaOH 0.01 N using phenolphtalein as indicator.

Results
1. Dark CO2 /ixation and temperature
Leaves of K. pinnata showed a reduction in total dark CO 2 fixation (integrated
over a 12 h dark period) with increasing leaf temperature (Table 1). Different leaf
temperatures however, did only slightly affect the maximal rate of dark CO2 fixation,
and the reduced total carbon gain at high temperature could be attributed to the delay
in attaining active CO2 fixation. The delay in the onset of net C02 dark fixation
increased the time necessary to reach maximum rate of C02 fixation. At a leaf tem-
perature of 12.7 °C net C02 fixation began a few minutes after onset of the dark peri-
od, while at 24.8 °C 3 hours elapsed until net C02 fixation commenced (Fig. 1).
Leaf conductance and, to a lesser extent, maximal and total rates of dark C02 fixa-
tion are reduced at high leaf-air vapor pressure deficits (VPD1-J over the temperature
range from 12-25 °C (Fig. 1). As a result, internal CO2 concentrations are lower at
low humidity (Fig. 2).

Z. P/lanzenphysioL Bd. 107. S. 251-258. 1982.

 Dark C02 fixation in K. pinnata 253

Table 1: Maximal rate and total dark C02 fixation of attached leaves of Kalanchoe pinnata.
Leaf temperature AverageVPD Maximal rate Total C02 fixation Time to reach
°C KPa JLmol C02 m- 2s- 1 mmol m- 2 nighC I max. rate
hours
12.7 0.13 2.86 97.1 5
16.7 0.10 2.89 78.5 7
21.7 0.20 2.49 58.4 10
24.8 0.20 2.45 40.3 10

Leaf temp "C VPD (KPo) Leaf temp.oC VPD (KPo) Leof temp. °C VPD (KPo)
127 013 16.7 010 24.8 022
()
30
122 075 16A 1.50 251 250 a
::J
Q

.,
U)
c:
a-
(")

::J
(")
'"
1 20 200 (j)
E
:3
a 150 :3
E ~
~ 1.0 100 :3 1

'"
U)
50 ...
1

a 24 4 22 24 4 6
20 22 6 20

TI M E
Fig. 1: Rate of dark CO 2 exchange and conductance to water vapor in attached leaves of
K. pinnata at different temperatures and humidities. C02 concentration of incoming air
335-360 I'll-I.

2. Leafair vapor pressure deficit and water use efficiency

The experiments conducted at high or low humidities with similar leaf tempera-
tures indicate that leaf stomatal conductance is more sensitive to VPD1_a than to
internal CO 2 concentration. The overwhelming effect of VPD1_a on leaf conduc-
tance can be seen in Fig. 3.
The ratio dark CO 2 fixationltranspiration at different temperatures and humidities
increased concomitantly with the increase in C02 fixation rate in the course of the
dark period (compare Fig. 1 and Fig. 4). In the low humidity treatments the ratio was
less than one half of the high humidity value, reflecting both the lower C02 influx
and the higher water loss. Towards the end of the dark period the CO 2/H20 ratio
tended to be similar at high and low temperatures, because maximal rate of dark C02
fixation at high temperature was reached during the second half of the dark period
(see Fig. 1).

Z. Pjlanzenphysiol. Ed. 107. S. 251-258. 1982.

254 ERNESTO MEDINA

.-
I
If)

N
I 200 300 ::>
E CD
(5 ~
E 150 n
E
N
o
OJ
0
c 100 200 ~
a o Conductance
-0
::>
-0
c \ VPD 0.75 KPa
0 50
u

18 20 22 24 2 4 6
TIME
Fig. 2: Intercellular CO 2 concentration (C0 2)i and conductance for water vapor at different
leaf-air vapor pressure deficits in attached leaves of K pinnata. Leaf temperature was 12.7 °C at
0.13 KPa and 12.2 °C at 0.75 KPa VPDl- a •
200

Leaf tempelOture ·C

, 160
.. 0-12.5·
• _ 16.5·
<f) .1 Q
0_21.5°
N • 25 0
..r

Eo e

.
80°· 0
0 120 6
g

.
E
E

u
OJ
80
0 '"
c
0
U

.,
::l 0
U
C 0
0
0
u
40
"""00 ;;

00
,~
It

008 QIO 0.20 040 0.60 080 1.00 2.00 3.00

Leaf- Air VPD (KPa )
Fig. 3: Relationship between conductance for water vapor and leaf-air vapor pressure deficit at
different temperatures in attached leaves of K pinnata.

Total acid accumulation during the dark period was reduced with increasing tem-
perature and high VPD1- a in a similar magnitude as the total dark C02 fixation.
Therefore, under all temperature and humidity treatments net amount of CO2 fixed
during the night was linearly correlated with the net amount of acid accumulated

Z. Pjlanzenphysiol. Bd. 107. S. 251-258. 1982.

 Dark C02 fixation in K. pinnata 255

Leaf temp. °C VPD KPa

~ 12.7 0.13
o 12.2 0.75
• 24.8 0.22
• 25.1 2.50
20

o 25
E
N
o
u
-g 10
E 50
()
75 o
N
!OO
150

4 6
TIME
Fig. 4: Ratio CO 2 uptake to transpiration (or the inverse transpiration ratio) in attached leaves
of K. pinnata at different temperatures and leaf-air vapor pressure deficits.

meq .acid ~ 3864 +198 m mol CO 2

240 r~0 .968*

E 200
0>
.C'
'l' 160
E
"0
U
0 120
:>
5
r::r /
Q) 80 /
E /
/
<l /

Fig. 5: Relationship between acid accumulation 20 40 60 80 100

(meq m- 2 nighC 1) and net CO2 fixation in at- m mol CO2 m-2 night- 1
tached leaves of K. pinnata.

(Fig. 5). The regression in Fig. 5 predicts an accumulation of 39 meq acid with zero
net C02 fixation, actual accumulation at 16 °C, measured in CO2 free air, was 28.6
meq acid. This value possibly reveals the amount of internal C02 (respiratory C02)
which is refixed and converted to organic acid, when no external C02 is available for
CAM activity.

z. Pjlanzenp/rysiol. Bd. 107. S. 251-258. 1982.

256 ERNESTO MEDINA

Discussion
The results presented in this paper confirm earlier reports on the sensitivity of sto-
mata of CAM plants to low humidity (Conde and Kramer 1975; Lange and Medina
1979; Osmond et al. 1979). At low VPDI_a (::c::: 0.20 KPa) stomatal conductance of
K. pinnata leaves reached high values at all temperatures tested, at least during the
second half of the dark period (Fig. 1 and 2). At high VPDI_a stomatal conductance
was strongly reduced, nevertheless dark C02 fixation rate reached high values close
to those observed at low VPD. Evidently, dark C02 fixation at low VPD proceeded
at a markedly decreased intercellular C02 concentration. Variations of stomatal con-
ductance during the night are inversely related to internal CO 2 concentration, how-
ever, results presented here indicate that stomata of K. pinnata are less sensitive to
internal CO 2 concentration at high VPDI _a (Fig. 3).
At high dark temperature two major factors might contribute to delay a net dark
CO2 fixation: a) a disturbance of carbohydrate metabolism which supplies PEP (Sut-
ton 1975); b) a higher permeability of the vacuole to malate (Llittge et al. 1975) there-
fore causing inhibition of PEP-carboxylase due to high malate content in the cyto-
plasm.
Inhibition of dark C02 fixation at high temperature is overcome as the dark period
proceeds, how it occurs can be only hypothesized. It is possible that for PEP-car-
boxylase to operate efficiently in the cell at temperatures above optimum a higher
PEP concentration is required, the delay observed would be the time needed to build
up this amount of PEP. Simultaneously the pronounced output of C02 observed at
the beginning of the dark period is attributed to increased respiration.
The linear relationship between CO 2 fixation and acid accumulation over a wide
temperature range confirm previous observations in K. daigremontiana (Medina and
Osmond 1981) and emphasizes further the relative minor role of respiration as endo-
genous CO 2 source. This relationship holds for high and low temperatures, with
fully open or semiclosed stomata. Present results with K. pinnata show that there was
always a higher proportion of acids accumulated than the predicted 2 : 1 ratio. This
deviation may be caused by the contribution of dark respiration to total acid accu-
mulation, but then it is not clear why the deviation is constant over a wide tempera-
ture range.
The ratio of protons accumulated! mol C02 fixation during the night has been
shown to be 2 : 1 in several CAM plants (Bjorkman and Osmond 1974; Medina and
Delgado 1976, Nobel and Hartsock 1978; O'Leary and Osmond 1980; Medina and
Osmond 1981). There are two notable exceptions, however. In Opuntia C02 fixation
accounted only for 40-55 % of the acid accumulated (Osmond et al. 1979). Further,
nocturnal acidification in drought stressed cacti with closed stomata seems
exclusively due to refixation of respiratory CO 2 (Szarek et al. 1973; Szarek and Ting
1974).
Experiments with K. pinnata in C02 free air not reported here, show also that

Z. Pjlanzenphysiol. Ed. 107. S. 251-258. 1982.

 Dark C02 fixation in K. pinnata 257

under conditions of strongly restricted CAM activity still a substantial acid accumula-
tion may take place; the most probable CO 2 source was respiration.
The interaction between CAM and respiratory processes might help explain the
kinetics of net dark CO 2 exchange at different temperatures. Morel (1979) has shown
that CAM activity may regulate substrate supply for respiration during the night.
Active CAM would therefore result in lower endogenous CO 2 production, and also
lower oxygen consumption. Several observations on the oxygen consumption by
CAM plants during the night show a tendency to a lower O 2 uptake rate when CO2
uptake is maximal (Brunhoffer et al. 1968 a, b; Kaplan et al. 1976; Andre et al. 1979).

Acknowledgements
I am most grateful to Prof. H. Mooney and his laboratory staff, specially George Koch, for
all the help received while using the gas exchange system at Stanford; to Elvira Cuevas for her
help in cultivating the plants and setting up the experiments. Thanks are also due to Dr. K.
Winter (University Wiirzburg, Germany) for his critical commentaries which improved the
presentation of this paper.

References
ANDRE, M., D. A. THOMAS, VON D. J. WILLERT, and A. GERBAUD: Oxygen and carbon dioxide
exchanges in Crassulacean-acid-metabolism-plants. Planta 147, 141-144 (1979).
BJORKMAN, O. and C. B. OSMOND: Effect of oxygen and carbon dioxide fixation in Kalanchoe
daigremontiana. Carnegie Year Book 72, 852-859 (1974).
BRANDON, D. c.: Temperature features of enzymes affecting crassulacean acid metabolism.
Plant Physiol. 42, 977-984 (1967).
BRUNNHOFER, H., H. SCHAUB und K. EGLE: Der Verlauf des CO 2- und 02-Gaswechsels bei
Bryophyllum daigremontianum in Abhangigkeit von der Temperatur. Z. Pflanzenphysiol.
59,285-292 (1968 a).
- - - Die Beziehungen zwischen den Veranderungen der Malat- und Starke-Konzentration
und dem C02- und 02-Gaswechsel bei Bryophyllum daigremontianum. Z. Pflanzenphysiol.
60,12-18 (1968 b).
CONDE, L. F. and D. J. KRAMER: The effect of vapor pressure deficit and diffusion resistance in
Opuntia compressa. Can. J. Bot. 53, 2923-2926 (1975).
COWAN, I.: Stomatal behavior and environment. Advances in Botanical Research, R. D. PRE-
STON and H. W. WOODHOUSE (eds.), Vol. 4, pp. 117-228 (1978).
GULMON, S. L. and A. J. BLOOM: C3 photosynthesis and high temperature acclimation of CAM
in Opuntia basilaris. Engelm. and Biogel. Oecologia (Berl.) 38,217-222 (1979).
KAPLAN, A.,J. GALE, and A. POLJAKOFF-MAYBER: Resolution of net dark fixation of carbon diox-
ide into its respiration and gross fixation components in Bryophyllum daigremontianum. J.
Exp. Botany 27,220-230 (1967).
KLUGE, M. and I. P. TING: Crassulacean acid metabolism. Ecological Studies 30. Springer
Verlag, Berlin, 1978.
LANGE, O. L. and E. MEDINA: Stomata of the CAM plant Tillandsia recurvata respond directly
to humidity. Oecologia (Berl.) 40,357-363 (1979).
LUTTGE, U., E. BALL, and H.-W. TROMBALLA: K+ independence of osmoregulated oscillations of
malateZ-levels in the cells of CAM leaves. Biochem. Physiol. Pflanzen 167, 267-283 (1975).
MEDINA, E. and M. DELGADO: Photosynthesis and dark C02 fixation in Echeveria columbiana v.
Poellnitz. Photosynthetica 10,155-163 (1976).

Z. Pjlanzenphysiol. Bd. 107. S. 251-258. 1982.

258 ERNESTO MEDINA

MEDINA, E. and C. B. OSMOND: Temperature dependence of dark CO2 fixation and acid accu-
mulation in Kalanchoe daigremontiana. Austr. J. Plant Physiology 8, 641-648 (1981).
MOREL, c.: Role coordinateur du CAM dans Ie metabolisme intermediaire. Physiol. Veg. 17,
697-712 (1979).
NOBEL, P. and T. HARTSOCK: Resistance analysis of nocturnal carbon dioxide uptake by a Cras-
sulacean Acid Metabolism succulent, Agave deserti. Plant Physiol. 61, 510-514 (1978).
O'LEARY, M. H. and C. B. OSMOND: Diffusional contribution to carbon isotope discrimination
during dark C02 fixation in CAM plants. Plant Physiol. 66, 931-934 (1980).
OSMOND, C. B., D. L. NOTT, and P. M. ZINTH: Carbon assimilation patterns and growth of the
introduced CAM plants Opuntia inermis in Eastern Australia. Oecologia 40,331-350 (1979).
SUTTON, B. G.: The path of carbon in CAM plants at night. Aust. J Plant Physiol. 2, 377-387
(1975).
SZAREK, S. R., H. B. JOHNSON, and I. P. TING: Drought adaptation of Opuntia basilaris: sig-
nificance of recycling carbon through crassulacean acid metabolism. Plant Physiol. 52,
539-541. (1973).
SZAREK, S. R. and I. P. TING: Physiological responses to rainfall in Opuntia basilaris. Am. J Bot.
62,602-609 (1974).
TING, I. P., M. L. THOMPSON, and W. M. DUGGER JR.: Leaf resistance to water vapor transfer in
succulent plants: effect of thermoperiod. Am. J Bot. 54, 245-251 (1967).
WINNER, W. E. and H. A. MOONEY: Ecology of S02 resistance: I. Effects of fumigations on gas
exchange of deciduous and evergreens. Oecologia (Berl.) 44, 290-295 (1980).
WOLF, J: Der diurnale Saurerhythmus. In: RUHLAND, W. (ed.), Encyclopedia of Plant Physiol.
Vol. 12(2), pp. 808-889 (1960).

Z. Pf/anzenphysiol. Bd. 107. S. 251-258. 1982.