Crassulacean acid metabolism in the ZZ plant, Zamioculcas zamiifolia (Araceae)

Abstract

Zamioculcas zamiifolia (Araceae), a terrestrial East African aroid, with two defining attributes of crassulacean acid metabolism (CAM) (net CO(2) uptake in the dark and diel fluctuations of titratable acidity) is the only CAM plant described within the Araceae, a mainly tropical taxon that contains the second largest number of epiphytes of any vascular plant family. Within the Alismatales, the order to which the Araceae belong, Z. zamiifolia is the only documented nonaquatic CAM species. Zamioculcas zamiifolia has weak CAM that is upregulated in response to water stress. In well-watered plants, day-night fluctuations in titratable acidity were 2.5 μmol H(+)·(g fresh mass)(-1), and net CO(2) uptake in the dark contributed less than 1% to daily carbon gain. Following 10 d of water stress, net CO(2) uptake in the light fell 94% and net CO(2) uptake in the dark increased 7.5-fold, such that its contribution increased to 19% of daily carbon gain. Following rewatering, dark CO(2) uptake returned to within 5% of prestressed levels. We postulate that CAM assists survival of Z. zamiifolia by reducing water loss and maintaining carbon gain during seasonal droughts characteristic of its natural habitat.

Full text

American
Journal
of
Botany
94(10):
1670-1676.
2007.
CRASSULACEAN
ACID
METABOLISM
IN
THE
ZZ
PLANT,
ZAMIOCULCAS
ZAMIIFOLIA
(ARACEAE)^
JOSEPH
A.
M.
HOLTUM,^-^
KLAUS
WINTER,^
MARK
A.
WEEKS,^
AND
TIMOTHY
R.
SEXTON^
^School
of
Marine
and
Tropical
Biology,
James
Cook
University,
Townsville,
Queensland
4811,
Australia;
and
^Smithsonian
Tropical
Research
Institute,
Balboa,
Ancón,
Republic
of
Panama
Zamioculcas
zamiifolia
(Araceae),
a
terrestrial
East
African
aroid,
with
two
defining
attributes
of
crassulacean
acid
metabolism
(CAM)
(net
CO2
uptake
in
the
dark
and
diel
fluctuations
of
titratable
acidity)
is
the
only
CAM
plant
described
within
the
Araceae,
a
mainly
tropical
taxon
that
contains
the
second
largest
number
of
epiphytes
of
any
vascular
plant
family.
Within
the
Alismatales,
the
order
to
which
the
Araceae
belong,
Z.
zamiifolia
is
the
only
documented
nonaquatic
CAM
species.
Zamioculcas
zamiifolia
has
weak
CAM
that
is
upregulated
in
response
to
water
stress.
In
well-watered
plants,
day-night
fluctuations
in
titratable
acidity
were
2.5
|.tmol
H+(g
fresh
mass)^',
and
net
CO2
uptake
in
the
dark
contributed
less
than
1%
to
daily
carbon
gain.
Following
10
d
of
water
stress,
net
CO2
uptake
in
the
light
fell
94%
and
net
CO2
uptake
in
the
dark
increased
7.5-fold,
such
that
its
contribution
increased
to
19%
of
daily
carbon
gain.
Following
rewatering,
dark
CO2
uptake
returned
to
within
5%
of
prestressed
levels.
We
postulate
that
CAM
assists
survival
of
Z.
zamiifolia
by
reducing
water
loss
and
maintaining
carbon
gain
during
seasonal
droughts
characteristic
of
its
natural
habitat.
Key
words:
Araceae;
CO2
exchange;
crassulacean
acid
metabolism;
drought
stress;
photosynthesis;
Zamioculcas.
Crassulacean
acid
metabolism
(CAM)
is
the
second
most
common
pathway
of
photosynthesis
in
vascular
plants
(Winter
and
Smith,
1996).
CAM
has
evolved
often,
with
species
distributed
in
29
families
and
338
genera
of
flowering
plants
(Smith
and
Winter,
1996;
Silvera
et
al,
2005;
Liu
and
Wang,
2006),
two
families
of
gymnosperms
(Vovides
et
al.,
2002;
von
Willert
et
al.,
2005),
two
families
of
leptosporangiate
ferns
(Hew
and
Wong,
1974;
Carter
and
Martin,
1994;
Holtum
and
Winter,
1999),
and
one
family
of
lycophytes
(Keeley,
1981).
Initially
considered
primarily
a
water-conserving
adaptation
of
terrestrial
succulent
plants
to
hot,
semi-arid
environments,
CAM
assists
species
in
a
diverse
range
of
habitats
(Skillman
et
al.,
2005).
The
majority
of
CAM
plants
are
probably
epiphytes
in
tropical
and
subtropical
forests
(Crayn
et
al.,
2004;
Holtum
and
Winter,
2005;
Silvera
et
al.,
2005),
but
CAM
has
also
been
reported
in
tropical
trees
(Gehrig
et
al.,
2003;
Holtum
et
al.,
2004;
Lüttge,
2006),
halophytes
(Winter
and
Holtum,
2005,
2007),
alpine
succulents
(Osmond
et
al.,
1975),
aquatic
plants
of
oligotrophic
lakes
or
seasonal
pools
(Keeley,
1981,
1996),
and
in
plants
without
stomata
that
obtain
CO2
via
their
roots
(Keeley
et
al.,
1984).
The
CAM
pathway,
which
enables
plants
to
successfully
live
in
such
a
range
of
environments,
involves
the
ability
to
fix
CO2
during
the
dark,
storing
the
carbon
as
malic
acid
in
vacuoles.
In
the
light,
the
malic
acid
is
decarboxylated,
and
the
CO2
evolved
is
refixed
by
Rubisco
and
used
for
growth
and
maintenance
(Holtum
et
al.,
2005).
CAM
species
with
functional
stomata
are
highly
water-use
efficient
because
decarboxylation
and
CO2
refixation
are
accompanied
by
reduced
stomatal
aperture
and,
consequently,
lower
rates
of
transpiration
(Winter
et
al.,
2005).
In
aquatic
plants,
CAM
is
an
adaptation
to
C02-limited
'
Manuscript
received
5
February
2007;
revision
accepted
6
August
2007.
The
authors
acknowledge
the
support
of
a
Magdalen
College
Visiting
Fellowship
(J.A.M.H.);
J.A.C.
Smith
and
the
Department
of
Plant
Sciences,
University
of
Oxford
(J.A.M.H.);
the
Andrew
W.
Mellon
Foundation
(K.W.);
and
the
Smithsonian
Tropical
Research
Institute
(K.W.
and
J.A.M.H.).
C.
Finney
(James
Cook
University,
Townsville)
provided
histological
support
and
T.
B.
Croat
(Missouri
Botanical
Garden)
and
C.
Galdames
(Smithsonian
Tropical
Research
Institute)
identified
Amhurium
michelii.
'^Author
for
correspondence
(e-mail:
joseph.holtum@jcu.edu.au)
environments
in
which
the
levels
of
dissolved
CO2
are
either
permanently
low
or
low
during
the
light
(Keeley,
1996).
Unlike
C3
or
C4
photosynthesis,
the
phenotypic
expression
of
CAM
is
not
an
all-or-nothing
phenomenon.
Some
CAM
species
obtain
virtually
all
of
their
CO2
during
the
dark,
most
species
obtain
CO2
during
the
dark
and
the
light,
and
in
some
species
dark
CO2
fixation
is
restricted
to
the
refixation
of
respiratory
CO2.
Such
variety
of
expression
increases
the
utilitarian
nature
of
CAM
(Osmond,
2007).
The
ecological
adaptability
of
CAM
plants
is
often
enhanced
by
an
ability
to
modify,
in
response
to
environmental
conditions,
the
relative
amounts
of
CO2
assimilated
in
the
light
via
the
less
water-use
efficient
C3
photosynthesis
vs.
CO2
assimilated
in
the
dark
by
the
more
water-use
efficient
CAM
process.
The
ability
to
express
CAM
may
be
constitutive
or
facultative,
developmen-
tally
programmed,
or
induced
in
response
to
stress.
The
majority
(>90%)
of
epiphytes
with
CAM
are
in
the
monocotyledonous
families
Bromeliaceae
(Poales)
and
Orchid-
aceae
(Asparagales).
In
1989,
Kress
calculated
that
of
an
estimated
23
466
species
of
vascular
plants
that
are
epiphytes
(16
610
monocots
and
magnoliids,
4253
eudicots,
four
gymnosperms,
and
2599
ferns
or
fern
allies;
roughly
10%
of
vascular
plants
in
toto),
74%
of
the
species
are
in
five
families:
the
Orchidaceae
(13
951
species),
the
Araceae
(1349
species),
the
Bromeliaceae
(1145
species),
the
Polypodiaceae
(1029
species),
and
the
Piperaceae
(710
species).
Though
CAM
is
well
represented
in
the
Orchidaceae
and
the
Bromeliaceae
and
moderately
represented
in
the
Piperaceae,
few
species
have
been
described
in
the
Polypodiaceae,
and
CAM
has
never
been
reported
in
the
Araceae
(Smith
and
Winter,
1996),
the
family
with
the
second
highest
number
of
epiphytic
species.
The
absence
of
documented
CAM
species
from
the
Araceae
is
surprising
because
it
is
large
(at
least
4025
species
and
106
genera;
Croat
[2004];
Stevens
[2006]),
mainly
tropical,
and
epiphyte-rich.
Moreover,
some
epiphytes,
particularly
in
the
genus
Anthurium,
sport
succulent
leaves
so
characteristic
of
plants
with
CAM.
Here
we
report
the
presence
of
CAM
in
a
terrestrial
aroid,
Zamioculcas
zamiifolia
Schott
(Araceae).
Apart
from
having
CAM,
Z.
zamiifolia
is
an
atypical
aroid
in
several
taxonomic
1670

October
2007]
HOLTUM
ET
AL.
CAM
PHOTOSYNTHESIS
IN
ZAMIOCULCAS
1671
TABLE
1.
Day-night
variation
in
titratable
acidity,
expressed
as
\imol
H+-(g
fresli
mass)^',
of
leaflets
from
well-watered
Zamioculcas
zamiifolia
in
comparison
to
two
C3
aroids,
Epipremnum
aureum
and
Anthurium
michelii,
and
known
CAM
species.
Values
are
the
means
of
three
replicates
(±SE)
with
the
exception
of
the
values
for
Platycerium
veitchii
for
which
N
=
5.
The
significance
of
dawn-dusk
differences
in
titratable
acidity
was
tested
using
nonpaired
one-tailed
t
tests.
Dusk
Dawn
Dawn-dusk
Photosynthetic
pathway
and
plant
species
Hmol
H+'(g
fresh
mass)"'
(±SE)
|j.mol
H"'"-(g
fresh
mass)"'
(±SE)
[imol
11+'(g
fresh
mass)"'
df
T
p
C3
Epipremnum
aureum
5.2
±
0.2
5.4
±
0.3
0.2
4
0.51
>0.05
Anthurium
michelii
6.8
±
0.9
6.6
±
0.2
-0.2
4
0.36
>0.05
CAM
Zamioculcas
zamiifolia
3.5
±
0.2
6.0
±
0.5
2.5
4
8.24
<0.05
Platycerium
veitchii'^
9.8
±
1.0
13.9
±
1.5
4.1
8
2.34
<0.05
Agave
attenuata
5.9
±
0.5
53.5
±
5.1
47.6
4
9.32
<0.05
Cissus
rotundifolia
18.5
±
5.0
111.4
±
7.4
93.0
4
10.46
<0.05
Kalanchoe
pinnata
11.6
±
2.2
152.6
±
18.5
141.0
4
5.69
<0.05
^
The
values
for
Platycerium
veitchii,
a
weak
CAM
plant,
are
from
Holtum
and
Winter
(1999)
and
are
included
for
comparison.
and
ecological
respects.
Within
the
Araceae,
Zamioculcas
is
a
monotypic
genus
in
the
Zamioculcadeae,
a
tribe
basal
to
the
subfamily
Aroideae
(Hesse
et
al.,
2001;
Tarn
et
al.,
2004;
Bogner
and
Hesse,
2005).
Unique
in
the
Araceae,
the
Zamioculcadeae
can
propagate
vegetatively
from
fallen
leaflets.
Not
a
wet
rainforest
species
like
most
aroids,
Z.
zamiifolia
inhabits
humid
to
seasonally
dry
forests,
open
bushland,
and
savannas
in
tropical
east
and
subtropical
southeast
Africa
(Peter,
1929;
Mayo
et
al.,
1997;
Newton,
1997),
where
it
is
seldom
found
above
800
m
a.s.l.
Its
ability
to
tolerate
water
stress
and
low
light
has
elevated
Z.
zamiifolia
to
international
horticultural
importance
(the
"ZZ
plant";
Chen
and
Henny,
2003).
Belowground,
plants
consist
of
a
large
tuber
and
a
short,
thick
underground
stem
from
which
arise
compound
leaves.
Aboveground,
each
compound
leaf
consists
of
4-8
pairs
of
oblong-elliptic,
glabrous,
coriaceous,
slightly
succulent
leaflets
borne
on
an
elongate
rachis
attached
to
a
succulent
petiole
(Mayo
et
al.,
1997).
cm
high
and
1.9
cm
diameter,
was
detached
from
the
stem
under
water
and
was
sealed
into
a
beaker
containing
water.
The
detached
petiole
and
beaker
were
placed
in
the
gas-exchange
system.
Extraction
and
assay
of
PEP
carboxylase
(PEPC)
•Deacidified
tissue,
frozen
in
liquid
N2,
was
extracted
in
10
volumes
of
ice-cold
buffer
containing
200
mM
tricine-KOH
pH
8.0,10%
(v/v)
ethanediol,
5
mM
EGTA,
5
mM
MgCh,
1%
(w/v)
BSA,
5
mM
dithiothreitol
(DTT),
0.1%
(v/v)
Triton
X-100,
and
insoluble
poly
vinylpolypyrrolidone
(PVPP)
of
a
mass
equal
to
that
of
the
tissue.
The
extracts
were
filtered
through
two
layers
of
cheesecloth,
centrifuged
at
120
000
X
g
for
10
min,
and
desalted
through
Sephadex
G-25
(PD-10
column;
GE
Life
Sciences,
NSW,
Australia)
with
grinding
medium
minus
Triton-100,
PVPP,
and
BSA.
PEPC
was
assayed
at
30°C
according
to
Kluge
et
al.
(1981).
Titratable
acidity
•Titratable
acidity
(|xmol
H+)
was
determined
by
measuring
the
volume
of
5
or
10
mM
NaOH
required
to
neutralize
to
pH
7.0
extracts
of
leaves
of
known
masses
that
had
been
frozen
in
liquid
N2
and
then
boiled
sequentially
in
50%
ethanol
and
water.
RESULTS
MATERIALS
AND
METHODS
Plant
material
•Plants
from
the
family
Araceae
and
known
CAM
species
from
other
plant
families
were
cultivated
in
pots,
with
regular
watering
and
periodic
provision
of
slow-release
complete
fertilizer
at
the
Smithsonian
Tropical
Research
Institute,
Panama
City,
Republic
of
Panama
[Anthurium
michelii
Guillaumin
(Araceae)
and
Z.
zamiifolia];
the
Department
of
Plant
Sciences,
Oxford,
UK
[A.
harrisii
(Graham)
G.
Don
(Araceae),
Kalanchoe
daigremontiana
Raym.-Hamet
&
H.
Perrier
(Crassulaceae)
and
Z.
zamiifolia};
and
the
School
of
Marine
and
Tropical
Biology,
James
Cook
University,
Australia
[Agave
attenuata
Salm-Dyck
(Agavaceae),
Cissus
rotundifolia
Vahl
(Vitaceae),
Epipremnum
aureum
(Linden
&
André)
G.
S.
Bunting
(Araceae),
Kalanchoe
pinnata
(Lam.)
Pers.
(Crassulaceae)
and
Z.
zamiifolia}.
Measurements
of
CO
2
exchange
•Net
CO2
exchange
by
Z.
zamiifolia
was
measured
for
distal
sections
of
compound
leaves
containing
eight
leaflets
and
associated
rachis.
For
A.
michelii,
a
single
attached
fully
expanded
leaf
was
measured.
Leaves,
still
attached
to
the
parent
plant,
were
sealed
with
Terostat
VII
(Henkel-Teroson,
Düsseldorf,
Germany),
inside
a
gas-exchange
cuvette
(GWK-3M,
Walz,
Effeltrich,
Germany)
in
a
controlled-environment
chamber
operating
under
12
h
light
(28°C,
350
nmol
photonm"2s"')/12
h
dark
(22°C)
cycles.
Dew
point
of
air
entering
the
chamber
was
18°C.
Net
CO2
exchange
was
measured
using
a
LI-6252
CO2
analyzer
(LI-COR,
Lincoln,
Nebraska,
USA)
in
a
flow-through
gas-exchange
system
(Holtum
and
Winter,
2003)
operating
at
2.38
L
airmin^'.
Air
was
sourced
16
m
above
ground
level
and
passed
through
a
1-m^
buffer.
The
CO2
exchange
of
a
succulent
petiole
of
Z.
zamiifolia
was
quantified
for
two
day-night
cycles
in
the
gas-exchange
system
described.
The
petiole,
4.9
Zamioculcas
zamiifolia
has
day-night
fluctuations
in
titratable
acidity
•Well-watered
Z.
zamiifolia
accumulated
H"*"
in
leaflets
during
the
dark
(Table
1).
In
comparison,
leaves
of
E.
aureum,
a
C3
aroid
climber,
and
A.
michelii,
an
epiphytic
aroid
with
thick
leaves,
did
not
accumulate
H"*"
during
the
dark.
The
day-night
fluctuations
of
H+
of
2.5
(imol
H+-(g
fresh
mass)"'
in
Z.
zamiifolia
were
small
compared
to
those
of
three
species
with
strongly
expressed
CAM
in
which
maximal
diel
acidity
changes
ranged
between
48
and
141
|imol
H+-(g
fresh
mass)"'.
PEPC
activity
from
Z.
zamiifolia
is
greater
than
in
two
C3
aroids
•In
a
pattern
similar
to
that
observed
for
H+
fluctuations,
the
extractable
activity
of
PEPC
from
Z.
zamiifolia
was
greater
than
the
activities
from
two
C3
members
of
the
Araceae,
8.5-fold
greater
than
that
from
E.
aureum
and
2.7-fold
greater
than
that
from
A.
harrisii,
but
was
only
one-eighth
that
of
a
strong-CAM
plant,
Kalanchoe
daigremomiana
(Table
2).
The
PEPC
activities
in
the
two
CAM
plants
differed
significantly
from
that
of
the
C3
species
(Mann-Whitney
test,
Z
=
2.88,
df=6.6,
P
<
0.01).
Well-watered
Z.
zamiifolia
has
net
CO2
uptake
in
the
dark
•Well-watered
Z.
zamiifolia
had
net
CO2
uptake
in
the
dark,
a
defining
characteristic
of
CAM
(Fig.
1).
The
small
amount
of
net
CO2
uptake
in
the
dark,
about
0.2%
ofthat
observed
in
the

1672
AMERICAN
JOURNAL
OF
BOTANY
[Vol.
94
TABLE
2.
Comparison
of
the
extractable
activity,
expressed
as
Hmol-min^'-(g
fresh
mass)^',
of
PEPC
from
three
members
of
the
Araceae
and
a
strong
CAM
plant,
Kalanchoe
daigremontiana.
Values
are
means
of
three
extractions
±
SE.
The
PEPC
activities
in
the
two
CAM
species
differed
significantly
(asterisks)
from
those
of
the
two
C3
species
(Mann-Whitney
test,
Z
=
2.88,
df
=
6.6,
P
<
0.01).
PEPC
activity
Taxon
jtmol-min"'-(g
fresii
mass)"'
Araceae
Zamioculcas
zamiifolia
(CAM)
Anthurium
harrisii
(C3)
Epipremnum
aureum
(C3)
Crassulaceae
Kalanchoe
daigremontiana
(CAM)
0.93
0.34
0.11
7.5
0.14*
0.06
0.04
0.43*
light,
was
insufficient
to
completely
offset
respiratory
CO2
lost
in
the
dark.
As
a
result,
carbon
balance
in
the
dark
was
negative
overall
(Fig.
2).
The
maximal
rate
of
CO2
uptake
in
the
light,
generally
observed
about
1
h
after
illumination
of
the
leaflets,
was
135-fold
greater
than
the
maximal
dark
rate,
which
occurred
3
to
4
h
after
the
onset
of
darkness.
13
14
15
Time
(days)
Fig.
1.
Net
CO2
exchange
by
Zamioculcas
zamiifolia
during
an
18-d
drying-rewetting
cycle.
Plants
were
grown
under
12
h
light
(28°C,
350
|.tmol
photon-m^^-s^')/12
h
dark
(22°C)
cycles.
Watering
ceased
on
day
3
and
was
reinitiated
after
the
onset
of
the
light
period
on
day
13.
Darkness
is
indicated
by
stippling,
watering
periods
by
closed
arrows,
and
drought
by
open
arrows.
0
O
O
O)
o
c
•
CO
O
O
•o
'S
g
OC
-2
-
200
100
0.16
¿
0.08
0.00
2
4
6
8
10
12
14
16
18
Time
(days)
Fig.
2.
(A)
CO2
balance
(squares)
and
net
CO2
uptake
(circles)
during
the
dark,
(B)
CO2
balance
in
the
light,
and
(C)
the
ratio
dark
:
light
CO2
balance
by
Zamioculcas
zamiifolia
during
the
18-d
drying-rewetting
cycle
shown
in
Fig.
1.
Watering
ceased
on
day
3
and
was
reinitiated
after
the
onset
of
the
light
period
on
day
13.
Watering
periods
are
indicated
by
closed
arrows
and
drought
by
open
arrows.
Net
CO2
uptake
in
the
dark
was
a
leaf
blade
phenomenon.
The
detached
petiole
had
net
CO2
loss
during
the
light
and
the
dark.
The
rate
of
CO2
loss
in
the
dark
averaged
0.5
|imolm~^-s"
(~0.08
^mol-kg-
less,
averaging
p,molkg~'s~').
•s
^),
whereas
in
the
light
the
loss
was
60%
approximately
0.2
(imolm~^s~'
(«=0.03
Dark
CO2
uptake
is
upregulated
by
water
stress
in
Z.
zamiifolia
•Following
the
imposition
of
water
stress,
carbon
gain
during
the
dark
increased
and
carbon
gain
during
the
light
decreased,
such
that
the
proportion
of
carbon
fixed
during
the
dark
rose
relative
to
the
light
(Fig.
2).
The
reduction
in
CO2
uptake
during
the
light
was
not
uniform.
Initially,
the
rate
of
CO2
uptake
decreased
late
in
the
light
period
but
not

October
2007]
HOLTUM
ET
AL.
CAM
PHOTOSYNTHESIS
IN
ZAMIOCULCAS
1673
TABLE
3.
Day-night
variation
in
titratable
acidity,
expressed
as
|j.mol
H+(g
fresh
mass)^',
in
leaflets
oí
Zamioculcas
zamiifolia
that
were
well
watered
and
then
grown
without
watering
for
10
d.
Plants
were
cultivated
in
a
growth
cabinet
under
conditions
described
in
the
Materials
and
Methods.
Values
are
the
means
of
four
replicate
leaves
(±SE).
Dawn
values
are
significantly
greater
(one-way
t
test)
than
dusk
values
for
well-watered
(df
=
6,
i
=
5.9,
P
<
0.001)
and
drought-treated
leaves
(df
=
6,
í
=
4.9,
P
<
0.001).
The
dawn-dusk
titratable
acidities
of
well-watered
and
drought-treated
leaves
differed
significantly
(df
=
6,
i
=
2.5,
P
<
0.05;
two-way
t
test).
Treatment
Dusk
^jnol
H+'Cg
fresh
mass)"
(±SE)
Dawn
^mol
H^'(g
fresh
mass)"
(±SE)
Dawn-dusli
|.imol
H^(g
fresh
mass)"
Zamioculcas
zamiifolia
Well-watered
Drought-treated
3.8
4.6
0.2
0.5
7.0
9.5
0.5
0.6
3.2
5.0
during
the
early
light
period.
Subsequently,
the
rates
of
uptake
in
the
early
light
period
also
decreased.
During
the
imposition
of
stress,
the
enhanced
carbon
gain
during
the
dark
resulted
from
higher
rates
of
dark
CO2
uptake
and
longer
periods
during
which
CO2
exchange
was
positive.
Net
carbon
exchange
during
the
dark
became
positive
after
1
d
without
watering
and
remained
so
for
the
10
d
without
watering.
Rewatering
of
drought-stressed
plants
was
accompanied
by
a
reduction
in
CO2
gain
during
the
dark,
which
was
initially
observed
during
the
first
night
after
rewatering,
and
an
increase
in
CO2
gain
during
the
light
(Figs.
1
and
2).
The
increase
in
dark
CO2
gain
in
response
to
drought
was
accompanied
by
a
56%
increase
in
H+
accumulation
in
comparison
to
well-watered
plants
(Table
3).
Anthurium
michelii
had
Cs-like
day-night
CO2
exchange
•
Anthurium
michelii,
an
epiphytic
Panamanian
rain-
forest
epiphyte
with
slightly
succulent
leaves
(leaf
thickness
of
0.50
±
0.01
mm
SE
in
comparison
to
0.70
±
0.01
SE
mm
forZ.
zamiifolia)
did
not
have
net
CO2
uptake
during
the
dark
during
2
d
under
well-watered
conditions
or
4
d
of
drought
(Fig.
3).
During
the
drought
treatment,
the
rate
of
dark
respiration
in
A.
michelii
fell
by
25%,
from
•0.12
to
•0.09
|.tmol-m~^s~'.
O
o
Fig.
3.
Net
CO2
exchange
by
Anthurium
michelii
during
a
6-day
wetting
and
drought
treatment.
The
plant,
grown
under
12
h
light
(28°C,
350
|j.mol
photon-m^^-s^')/12
h
dark
(22°C)
cycles,
was
not
watered
after
day
2.
Darkness
is
indicated
by
stippling,
watering
periods
by
closed
arrows,
and
drought
by
open
arrows.
DISCUSSION
Z.
zamiifolia
has
CAM
•The
essential
criteria
that
define
CAM
include
an
ability
to
fix
CO2
during
the
dark
and
to
store
the
carbon
fixed
in
the
vacuole
as
an
organic
acid,
generally
malic
acid.
Both
well-watered
and
water-stressed
Z.
zamiifolia
had
net
CO2
uptake
during
the
dark
and
day-night
fluctuations
in
titratable
acidity
(Fig.
1,
Table
1).
Thus,
Z.
zamiifolia
can
be
classified
as
a
CAM
plant.
Within
the
Araceae,
it
is
the
only
CAM
species
yet
reported,
and
within
the
Alismatales
it
is
the
only
documented
terrestrial
species
with
CAM.
CAM
in
Z.
zamiifolia
is
upregulated
in
response
to
water
stress
•The
expression
of
CAM
increased
in
response
to
water
stress.
After
only
2
d
without
watering,
both
the
rate
and
extent
of
dark
CO2
uptake
increased
such
that
CO2
balance
in
the
dark
shifted
from
negative
to
positive.
After
10
d
without
watering,
net
dark
CO2
uptake
increased
7.5-fold
(Figs.
1
and
2).
Although
CO2
uptake
in
the
dark
increased
in
response
to
water
stress,
the
overall
response
to
the
10
d
drought
treatment
was
a
reduction
by
93%
of
the
total
day-night
CO2
gain
(Figs.
1
and
2).
This
decrease
in
CO2
balance
involved
a
94%
reduction
in
CO2
gain
during
the
light
that
was
offset
by
a
small
increase
in
CO2
uptake
in
the
dark.
Initially,
the
reduction
in
CO2
uptake
in
the
light
was
confined
to
midday
and
afternoon
CO2
fixation,
but
CO2
uptake
during
the
morning
began
to
decrease
after
4
d
of
water
stress.
The
stimulation
of
CAM
in
response
to
water
stress
of
Z.
zamiifolia
was
reversible
(Figs.
1
and
2).
Upon
rewatering,
CO2
uptake
during
the
light
reverted
to
its
prestress
levels
and
dark
CO2
uptake
was
reduced,
with
net
CO2
uptake
in
the
dark
decreasing
by
95%
and
CO2
balance
in
the
dark
changing
from
positive
to
negative.
The
increase
in
net
CO2
uptake
in
the
dark
that
accompanied
drought
was
most
likely
not
solely
due
to
an
increase
in
PEPC-
catalyzed
uptake.
The
56%
greater
diel
fluctuation
in
titratable
acidity
in
the
water-stressed
plants
was
consistent
with
an
increase
in
net
flux
of
carbon
from
CO2
to
malic
acid,
but
the
rise
was
less
than
the
7.5-fold
predicted
from
measurements
of
net
dark
CO2
uptake
(Fig.
2;
Table
3).
Although
fluctuations
in
titratable
acidity
were
not
measured
for
the
leaves
inside
the
gas-exchange
cuvette
(they
were
measured
in
leaves
from
a
companion
plant
that
grew
in
the
growth
chamber),
it
is
probable
that
not
all
of
the
net
increase
in
carbon
flux
into
the
plant
was
sequestered
in
organic
acid.
A
component
of
the
net
increase
in
CO2
flux
into
the
water-stressed
tissue
in
the
dark
may
have
been
the
result
of
a
reduction
in
respiratory
carbon
loss
rather
than
increased
assimilation
per
se.
Stress-related

1674
AMERICAN
JOURNAL
OF
BOTANY
[Vol.
94
decreases
in
CO2
uptake
in
the
light
are
often
accompanied
by
reductions
in
respiratory
CO2
loss
in
the
dark,
a
phenomenon
exemplified
by
A.
miche
Hi,
which
has
a
25%
reduction
in
rates
of
CO2
loss
in
the
dark
after
4
d
without
water
(Fig.
3).
It
is
also
possible
that
some
of
the
CO2
fixed
in
the
dark
may
have
been
converted
to
nonacidic
metabolites.
However,
the
major
routes
by
which
the
early
products
of
PEPC-mediated
dark
CO2
uptake
are
metabolized
involve
passage
through
the
Krebs
cycle,
which
results
in
the
loss
of
the
CO2
originally
assimilated
(Holtum
et
al.,
2005).
We
conclude
that
the
net
transfer
of
dark-
fixed
carbon
to
non-acid
components
is
likely
to
be
small.
Zamioculcas
zamiifolia
is
a
weak
CAM
plant.
The
fluctua-
tions
in
titratable
acidity
of
3-5
p,mol
H+(g
fresh
mass)"',
the
extractable
activity
of
PEPC,
and
the
carbon
gain
in
the
dark
were
all
low
in
comparison
to
levels
in
well-documented
strong
CAM
species
(Fig.
2;
Tables
1-3).
Why
call
Z.
zamiifolia
a
CAM
plant
when,
under
well-watered
conditions,
it
obtains
0.2%
of
its
daily
carbon
during
the
dark,
and
even
when
stressed
and
CO2
uptake
in
the
dark
increased
7.5-fold
in
response
to
stress,
dark
CO2
uptake
was
only
1.5%
of
CO2
uptake
in
the
light
under
well-watered
conditions?
Zamioculcas
zamiifolia
could
be
categorized
as
a
C3
plant
with
a
small
capacity
for
CAM
if
the
term
CAM
is
applied
in
the
narrow
way
that
the
terms
C3
and
C4
are
generally
employed,
to
simply
describe
the
major
pathway
of
photosynthesis
by
which
the
plant
gains
carbon
throughout
its
lifetime.
However,
no
plant
species
known
is
exclusively
CAM,
and
the
expression
of
CAM
is
rarely
constant
throughout
the
developmental
and
environmen-
tal
lifetime
of
a
plant.
As
a
result
of
this
flexibility
of
expression
of
CAM,
the
current,
widely
used
definition
of
a
CAM
phenotype
is
a
plant
that,
at
any
time
during
its
lifecycle,
irrespective
of
the
amount
of
CO2
uptake
in
the
light,
assimilates
CO2
in
the
dark
and
temporarily
stores
the
carbon
as
organic
acids
in
the
vacuole,
thus
having
diel
fluctuations
in
titratable
acidity.
Ultimately,
the
presence
or
absence
of
the
CAM
cycle
must
be
defined
at
the
molecular
level.
There
is,
as
yet,
no
consensus
of
what
are
the
fundamental
molecular
markers
of
CAM.
Because
most
components
of
the
CAM
cycle
are
also
functionally
present
in
C3
plants,
the
most
promising
candidates
for
markers
are
CAM-specific
isogenes,
the
best
studied
of
which
is
PEPC
(Taybi
et
al.,
2004;
Gehrig
et
al.,
2005).
However,
because
CAM
appears
to
have
evolved
often,
it
will
be
necessary
to
verify
that
marker
isogenes
are
expressed
in
species
across
diverse
taxa.
What
is
the
advantage
of
CAM
for
Z.
zamiifolia?
•
Although
information
on
the
ecology
of
Z.
zamiifolia
is
scarce,
it
appears
that
in
east
Africa
Z.
zamiifolia
is
a
plant
of
rocky,
partially
shaded
sites
in
habitats
subject
to
prolonged
seasonal
drought
(Mayo
et
al.,
1997;
Newton,
1997).
Zamioculcas
zamiifolia
in
its
natural
habitat
of
high
evaporative
demand
probably
has
a
positive
dark
CO2
balance
throughout
much
of
the
year
as
long
as
leaves
are
present.
In
situ
water
stress
is
likely
to
be
more
prolonged
and
severe
than
that
imposed
by
us
experimentally.
In
our
experiments,
the
decrease
in
CO2
uptake
during
the
light
had
not
stabilized
after
10
d
of
water
stress
(Figs.
1
and
2)
and
could
be
expected
to
fall
further.
At
times,
the
contribution
of
CAM
to
24-h
carbon
gain
in
Z.
zamiifolia
in
its
natural
habitat
is
thus
likely
to
exceed
the
19%
we
observed.
The
role
of
CAM
in
the
survival
of
Z.
zamiifolia
is
that
it
contributes
to
the
maintenance
of
a
positive
carbon
balance
while
the
plant
reduces
water
loss
by
reducing
CO2
uptake
in
the
light.
Water-use
efficiency
is
thereby
increased,
and
the
period
of
net
carbon
gain
is
prolonged.
Eventually,
if
stress
persists,
the
rachis
and
leaflets
abscise,
reducing
the
above-
ground
plant
to
an
apparently
dormant
cluster
of
erect,
succulent,
petiole
bases
(Newton,
1997).
CAM
is
not
the
only
water-conserving
feature
of
Z.
zamiifolia.
Radiative
load
and
transpirational
water
loss
are
reduced
by
erect,
coriaceous,
reflective,
succulent
leaflets
with
abaxial
stomata,
and
by
the
erect,
cylindrical,
succulent
petiole
and
rachis.
The
leaflets
and
tuber
are
rarely
subject
to
herbivory,
an
observation
consistent
with
the
presence
of
chemical
defenses,
a
feature
common
in
the
Araceae
(Dring
et
al.,
1995).
Why
is
CAM
apparently
rare
in
the
Araceae,
particularly
in
epiphytes?
•CAM
is
a
derived
condition
that
has
evolved
repeatedly
in
response
to
selection
imposed
by
dry
conditions
and/or
selection
driven
by
low
levels
of
dissolved
carbon
(Griffiths,
1989;
Keeley,
1998).
CAM
is
uncommon
in
the
basal
angiosperms,
where
it
is
present
only
in
the
most
derived
order,
the
Piperales
(Peperomia
spp.;
Holthe
et
al,
1992).
Similarly,
in
the
basal
monocots,
CAM
is
absent
(Acórales,
Petrosaviales)
or
rare
(Alismatales).
Many
species
in
the
basal
monocots,
particularly
in
the
Alismatales,
the
order
in
which
the
Araceae
are
located,
inhabit
aquatic,
marine,
or
mesic
swampy
areas.
It
is
therefore
perhaps
not
surprising
that
both
known
CAM
species
in
the
Alismatales,
Sagittaria
suhulata
(L.)
Buchenau
(Alismataceae)
and
Vallisneria
americana
Michx.
(Hydrocharitaceae),
are
aquatic
(Keeley,
1981,
1996;
Webb
et
al.,
1988).
Fossil
Araceae
are
associated
with
lacustrine
deposits
and
generally
moist
habitats
(Friis
et
al.,
2004;
Wilde
et
al.,
2005).
Indeed,
the
Araceae
have
not
radiated
extensively
into
water-
limited
environments
(Mayo
et
al,
1997).
The
few
Araceae
that
inhabit
deserts
and
semiarid
regions,
or
grow
at
high
altitude,
tend
to
be
geophytes
that
are
characteristically
deciduous
or
seasonally
dormant.
Modem
Araceae
are
most
abundant
and
diverse
in
the
humid
tropics.
The
epiphytic
Araceae
are
principally
wet
forest
species,
generally
inhabiting
sites
less
exposed
than
those
of
most
bromeliads
and
orchids.
Benzing
(1989)
notes
that
although
many
Araceae
are
secondary
hemiepiphytes,
a
life
form
that
might
be
expected
to
be
well
adapted
to
intermittent
water
stress,
they
characteristically
exhibit
fewer
epiphytic
specializations
for
coping
with
water
stress
than
do
bromeliads
and
orchids.
As
a
result,
they
are
typically
restricted
to
more
moist
habitats
where
selection
pressure
for
CAM
may
not
be
as
pronounced.
If
ancient
Araceae
and
Isoetes,
the
oldest
lineage
of
plants
with
CAM,
were
plants
of
wetlands,
why
is
CAM
common
in
extant
Isoetes
but
not
in
the
Araceae?
A
selection
pressure
for
the
retention
of
CAM
in
Isoetes
may
have
been
their
poor
ability
to
compete
with
faster-growing
radiating
flowering
plants
(Keeley,
1998).
Presumably
by
retaining
CAM,
Isoetes
could
reduce
competition
for
inorganic
carbon
and
thus
could
grow
in
the
presence
of
more
vigorous
species
in
temporally
carbon-infertile
lakes.
CAM
also
provided
a
mechanism
for
survival
in
permanently
carbon-infertile
waters
and
enabled
them
to
utilize
locally
high
concentrations
of
CO2
in
muds.
In
contrast,
the
more
rapid
growth
rates
of
the
Araceae
may
have
remained
to
be
associated
with
C3
because
they
gleaned
sufficient
inorganic
carbon
in
lacustrine
environments.
Pre-
sumably
the
lacustrine
Araceae
adopted
amphibian
habits
that
provided
access
to
atmospheric
CO2.
The
selection
pressures
for
CAM
in
terrestrial
plants
living
in
extremely
moist
soils
were
unlikely
to
be
strong.
It
may
be
relevant
that
many
extant

October
2007]
HOLTUM
ET
AL.
CAM
PHOTOSYNTHESIS
IN
ZAMIOCULCAS
1675
TABLE
4.
List
of
20
orders
and
34
families
in
which
CAM
species
have
been
reported.
The
Ust
is
based
upon
that
of
Smith
and
Winter
(1996),
which
has
been
updated
using
the
phylogeny
compiled
by
Stevens
(2006).
Superscripts
denote
families
in
which
CAM
has
been
detected
since
Smith
and
Winter
(1996).
Phylum
(group)
Oider
Family
Lycopodiophyta
Isoetales
Isoetaceae
Pteridophyta
Polypodiales
Polypodiaceae
Vittariaceae
Cycadophyta
Cycadales
Zamiaceae^
Gnetophyta
Gnetales
Welwitschiaceae
Magnoliophyta
Magnoliids
Piperales
Piperaceae
Monocotyledons
Alismatales
Araceae''
Hydrocharitaceae
Alismataceae
Asparagales
Orchidaceae
Asphodelaceae
Agavaceae
Ruscaceae
Poales
Bromeliaceae
Commelinales
Commelinaceae
Eudicotyledons
Caryophyllales
Aizoaceae
Cactaceae
Portulacaceae
Didiereaceae
Saxifragales
Crassulaceae
Vitales
Vitaceae
Geraniales
Geraniaceae
Malpighiales
Passifloraceae
Clusiaceae
Euphorbiaceae
Oxalidales
Oxalidaceae
Cucurbitales
Cucurbitaceae
Gentianales
Rubiaceae
Apocynaceae
Lamíales
Lamiaceae
Gesneriaceae
Plantaginaceae
Apiales
Apiaceae
Asterales
Asteraceae
^
Vovides
et
al.
•^
This
study.
(2002)
lacustrine
Isoetes
are
amphibious
species
in
which
the
submerged
leaves
express
CAM
but
the
emerged
leaves
are
C3
(Keeley,
1996,
1998).
It
is
also
possible
that
CAM
may
be
more
common
in
the
Araceae
than
we
are
aware.
Certainly,
Araceae
are
often
under-
represented
in
isotopic
surveys
of
epiphytes
(Winter
et
al.,
1983;
Eamshaw
et
al.,
1987;
Carter
and
Martin,
1994),
possibly
because
the
surveyors
tend
to
collect
species
that
have
recognizably
succulent
leaves
and
tend
to
ignore
hemi-
epiphytes.
The
most
likely
araceous
candidates
for
CAM,
on
the
basis
of
epiphytic
habitat
and
succulence
of
leaves,
are
in
the
sections
Leptanthurium
and
Porphyrochitonium
of
the
genus
Anthurium
(subfamily
Pothoideae),
which
contains
over
800
species
(Mayo
et
al.,
1997).
To
date,
the
few
Anthurium
spp.
examined
in
carbon
isotope
surveys
have
Cs-like
S'^C
values,
e.g.,
nine
Anthurium
species
on
Barro
Colorado
Island,
Panama
had
S^^C
values
between
•28.1%o
and
•33.7%o
(Zotz
and
Ziegler,
1997).
However,
carbon
isotope
surveys
that
measure
the
integrated
day-night
carbon
uptake
signal
over
the
life
of
the
organ
sampled
rarely
have
the
resolution
required
to
detect
weak
CAM
in
plants
that
acquire
only
a
small
proportion
of
their
carbon
during
the
dark
(Winter
and
Holtum,
2002).
How
prevalent
is
CAM
in
vascular
plants?
•The
most
recent
estimate
of
the
prevalence
of
CAM
in
vascular
plants
is
that
of
Smith
and
Winter
(1996),
who
documented
CAM
in
33
families
and
328
genera
containing
about
16000
species.
The
species
number
was
based
on
the
estimation
that
50%
of
tropical
epiphytic
orchids
and
bromeliads
are
CAM.
The
1996
census
requires
updating
to
accommodate
reassessments
of
vascular
plant
taxonomy
(e.g.,
Asclepiadaceae
are
now
in
the
Apocynaceae,
and
Dracaenaceae
are
now
in
the
Ruscaceae;
Chase
et
al.,
2000;
Stevens,
2006),
the
report
of
CAM
in
a
cycad
(Vovides
et
al.,
2002),
an
extensive
survey
of
bromeliad
carbon
isotope
compositions
(Crayn
et
al.,
2004),
the
discovery
of
new
CAM
genera
in
the
Crassulaceae
and
Orchidaceae
(Silvera
et
al.,
2005;
Liu
and
Wang,
2006),
and
this
communication
of
CAM
in
the
Araceae.
We
report
that
CAM
is
present
in
34
families
and
343
genera
of
vascular
plants
(Table
4).
The
great
uncertainty
is
still
the
total
number
of
CAM
species.
An
improved
estimate
of
the
number
of
species
requires
extensive
carbon
isotope
surveys
of,
in
particular,
the
orchids.
We
suspect,
however,
that
there
are
many
species
like
Z.
zamiifolia,
in
which
the
contribution
of
dark
CO2
uptake
to
daily
carbon
gain
is
so
small
that
it
would
not
produce
an
identifiable
isotopic
signal
(Winter
and
Holtum,
2002;
Silvera
et
al.,
2005).
Discovery
of
these
CAM
species
requires
a
more
labor-intensive
quantification
of
H+
fluctuations.
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