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Effect of chemical control of stomata on transpiration and photosynthesis

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Proceedings
of
the
NATIONAL
ACADEMY
OF
SCIENCES
Volume
48
*
Number
7
*
July
15,
1962
EFFECT
OF
CHEMICAL
CONTROL
OF
STOMATA
ON
TRANSPIRATION
AND
PHOTOSYNTHESIS*
BY
ISRAEL
ZELITCH
AND
PAUL
E.
WAGGONER
DEPARTMENTS
OF
BIOCHEMISTRY
AND
OF
SOILS
AND
CLIMATOLOGY,
THE
CONNECTICUT
AGRICULTURAL
EXPERIMENT
STATION,
NEW
HAVEN
Communicated
by
H.
B.
Vickery,
May
16,
1962
The
water
transpired
from
leaves
and
the
CO2
fixed
in
them
are
assumed
to
pass
mainly
through
the
microscopic
pores,
the
stomata,
that
provide
a
ready
path
for
diffusion
of
gases
between
the
surrounding
atmosphere
and
the
leaf
interior.
It
was
recently
reported1
that
many
enzyme
inhibitors
close
stomata
in
the
light,
when
they
are
normally
open,
and
the
relative
closure
by
different
compounds
was
evaluated
by
a
leaf
disk
assay.
An
effective
substance
of
this
kind,
which
brings
about
closure
of
stomata
when
sprayed
on
whole
leaves,
has
now
been
found.
We
report
here
the
relation
between
the
results
obtained
by
the
leaf
disk
assay
and
those
obtained
by
foliar
sprays
on
intact
plants.
The
lack
of
translocation
of
the
compound
within
the
tissues
and
the
long
duration
of
the
induced
stomatal
closure
are
also
shown.
The
induced
closure
permitted
a
demonstration
of
the
relation
between
aperture
and
both
transpiration
and
C02
assimilation,
and
also
indicated
that
the
diffusion
rate
is
a
limiting
factor
in
these
processes.
Relation
between
Standard
Leaf
Disk
Assay
and
Effect
of
Foliar
Spray.-In
the
previous
work,
leaf
disks
were
floated
on
solutions
of
various
metabolic
inhibitors,
and
the
dimensions
of
the
stomata
were
subsequently
measured.
If
this
rapid
assay
method
is
to
be
used
as
an
indicator
of
field
performance,
it
must
be
correlated
with
the
effect
of
spraying
these
compounds
on
a
number
of
species
of
plants.
A
conservative
test
of
the
correlation
was
made
with
three
compounds
of
known
ability
to
close
stomata.
The
results
(Table
1)
show
that
similar
concentrations
are
effective
in
the
disk
assay
and
as
foliar
spray.
Another
test
of
the
correlation
was
made
by
assaying
and
spraying
the
same
compound
on
different
species.
In
a
standard
disk
assay.
3.3
X
10-5
M
phenyl-
mercuric
acetate
closed
15
per
cent
of
tobacco
and
87
per
cent
of
maize
stomata.
When
this
compound
was
applied
as
a
spray
at
a
concentration
of
10-4
M,
the
stomata
were
closed
to
the
extent
shown
in
Tables
2
and
3.
Thus
the
standard
leaf
disk
assay
can
be
employed
with
different
subclasses
of
plants
and
is
an
ad-
vantageous
means
of
discovering
useful
compounds.
Translocation
and
Duration
of
Effect.-The
use
of
phenylmercuric
acetate
was
sug-
gested
to
us
by
the
report2
that
phenylmercuric
chloride,
sprayed
on
tomato
and
potato
foliage,
diminishes
transpiration
and
increases
growth.
Phenylmercuric
1101
1102
BOTANY:
ZELITCH
AND
WAGGONER
PROC.
N.
A.
S.
TABLE
1
COMPARISON
OF
THREE
COMPOUNDS
IN
LEAF
DISK
ASSAYS
AND
AS
SPRAYS
ON
TOBACCO
LEAVES
Percentage
of
Stomata
Closed
Compound
Concentration
(M)
Disk
assay
Leaf
spray
Phenylmercuric
acetate
10
X
10-5
86
80
3.3
X
10-5
28
33
8-Hydroxyquinoline
10
X
10-i
30
54
1.0
X
10-4
7
Sodium
c-hydroxydecanesulfonate
10
X
10-4
64
41
5.0
X
10-4
31
In
the
standard
disk
assay,
disks
from
leaves
kept
in
the
dark
were
floated
on
water
(control)
or
on
the
solu-
tion
to
be
tested.
The
disks
were
illuminated
and
maintained
at
250
for
90
min
when
impressions
in
silicone
rubber
were
made.
The
proportion
closed
was:
100
less
the
ratio
of
the
proportion
of
stomata
at
least
2/,
wide
in
the
treated
disks
to
the
proportion
of
stomata
in
the
control
disks
at
least
2;&
wide.
Normally,
80-100%
of
the
sto-
mata
in
the
control
were
open.
The
a-hydroxysulfonate
was
dissolved
in
potassium
acetate
or tartrate
buffer
(0.005
M)
at
pH
4.4.
On
a
sunny
day,
upper
surfaces
of
individual
tobacco
leaves
on
a
single
plant
were
sprayed
with
the
solutions
(all
contained
0.02%
Triton
X-100)
and
control
leaves
were
sprayed
with
water
or
buffer
solution.
Later,
the
entire
plant
was
covered
with
a
transparent
plastic
bag
to
raise
the
enrivonmental
humidity,
and,
about
4
hr
after
spraying,
leaf
impressions
were
made.
TABLE
2
PERCENTAGE
OF
HAVANA
SEED
TOBACCO
STOMATA
OPEN
AFTER
SPRAYING
THE
LEAVES
WITH
PHENYLMERCURIC
ACETATE
Day
5
-
Day
14--
Leaf
Surface
Treatment
Plant
A
Plant
B
Plant
A
Plant
B
1
U
0
98
100
100
100
L
0
100
100
100 100
2
U
10-5
M
88
84
76
96
3
U
10-4
M
36
32
44
24
L
0
100
100
100
100
4
U
10-4
M
36
28
48
20
{L
10-4
M
70
76
80
60
In
the
greenhouse,
four
leaves
on two
plants
were
chosen
at
random
and
sprayed
on
the
upper
(U)
or
lower
(L)
surface
with
0,
10-0
or
10-4
M
phenylmercuric
acetate
in
0.02%
Triton
X-100.
The
plants
then
stood
for
5
and
for
14
days.
About
two
hr
before
impressions
were
made
on
clear
days,
the
plants
were
covered
with
a
plastic
bag
to
raise
the
environmental
humidity.
TABLE
3
PERCENTAGE
OF
MAIZE
STOMATA
OPEN
AFTER
SPRAYING
WITH
PHENYLMERCURIC
ACETATE
Day5
.-
Day
14
Plant
Plant
Leaf
Surface
Treatment
A
B
C
D
A
B
C
D
1
U
0
88
88
84
86
98
92
88
96
L
0
94
92
100
80
94
100
78
94
2
U
10-5
M
76
86
100
84
3
U
10-4M
66
70
68
12
L
0
94
94
98
44
4
U
10-4
M
66
62
80
2
L
10-4
M
88
44
84
58
In
the
greenhouse,
4
leaves
were
chosen
at
random
on
4
maize
plants.
They
were
sprayed
on
the
upper
(U)
or
lower
(L)
surface
with
0,
10-5
or
10
-4M
phenylmercuric
acetate
in
0.1%
Triton
X-100.
Subsequent
procedure
was
as
in
Table
2.
acetate
is
more
convenient
than
the
chloride
because
of
its
equal
effectiveness
and
greater
solubility
in
water.
Mercuric
chloride
and
acetate
are
ineffective
in
the
standard
disk
assay.
Presently,
phenylmercuric
acetate
is
our
most
effective
compound
and
was
employed
in
the
remaining
experiments
of
this
paper.
Tables
2
and
3
show
that
the
effect
of
this
substance
sprayed
on
leaves
is
observed
only
on
the
surface
treated.
Obviously,
the
substance
is
not
translocated
even
from
one
surface
of
the
leaf
to
the
other.
However,
the
stomata
of
the
upper
surface
of
tobacco
and
maize
leaves
are
more
readily
closed
than
those
of
the
lower
surface.
Furthermore
the
effect
is
a
prolonged
one.
At
least
14
days
after
the
VOL.
48,
1962
BOTANY:
ZELITCH
AND
WAGGONER
1103
leaves
of
tobacco
or
maize
were
sprayed
with
a
solution
as
dilute
as
10-4
M,
the
stomata
were
still
closed.
Effect
of
Stomatal
Closing
upon
Transpiration
and
Photosynthesis.-The
role
of
stomatal
opening
in
transpiration
and
photosynthesis
can
be
analyzed
in
terms
of
Fick's
first
law
of
diffusion:
T
=
D(Xf
-
Xp)I(L
+
S);
(1)
P
=
D'(X'f-
X'a)/(L
+
S
+
M).
(2)
T
and
P,
in
gm
cm-2
sec'-,
are
rates
of
transpiration
and
photosynthesis
per
unit
of
leaf
surface,
and
D
and
D',
in
cm2
sec-',
are
the
coefficients
of
diffusion
of
H20
and
C02
in
air.
The
Xf
and
Xp,
in
gm
cm-3,
are
the
concentrations
of
H20
in
the
surrounding
air
and
at
the
evaporating
surface,
i.e.,
the
surfaces
of
spongy
parenchymatous
cells
about
the
substomatal
cavities.
The
X',
and
X'a
are
the
concentrations
of
C02
in
the
free
air
and
at
the
site
of
an
acceptor
within
the
chloroplasts.
The
L
and
S
are
apparent
path
lengths
for
the
diffusion
in
the
surrounding
atmos-
phere
and
through
the
stomata.
These
terms
do
not,
of
course,
correspond
to
any
actual
lengths.
Rather,
L/D
and
S/D
represent
the
resistance
of
the
atmosphere
and
of
the
stomata
and
substomatal
cavities
to
the
diffusion
of
water.
The
M
is
the
apparent
length
of
the
aqueous
path,
which
encompasses
the
surface
of
the
mesophyll
cells
about
the
substomatal
cavities,
to
the
sites
of
the
C02
assimilation;
hence,
the
M
appears
in
the
equation
defining
the
diffusion
of
C02
in
photosynthesis,
but
not
in
that
for
transpiration.
The
usefulness
in
botany
of
equations
(1)
and
(2)
has
often3-5
been
demonstrated.
We
shall
use
it
both
to
relate
transpiration
and
photosynthesis
to
stomatal
aperture,
and
to
predict
the
relative
changes
in
transpiration
and
C02
assimilation.
We
may
calculate
S
from
observations
of
stomatal
width,
a.
We
chose
Penman
and
Schofield's4
relation
between
S
and
a:
S
=
[l/(7rab)
+
1/(2/ab)]
+
n,
(3)
where
n
is
number
of
stomata
per
cm2
(12,000
per
cm2
of
leaf
surface
for
tobacco),
1
is
depth
of
stomatal
opening
(1Ou)
and
b
is
length
of
aperture
(20,u).
The
mean
number
and
width
were
obtained
from
both
leaf
surfaces.
In
a
constant
environment,
equation
(1)
can
be
rewritten
as
a
regression
equation
1/T
=
constant
+
constant'(S),
(4)
where
S
is
estimated
from
observations
of
stomatal
width
and
equation
(3).
In
the
same
way,
1/P
can
be
related
linearly
to
S.
The
easy
demonstration
of
the
effect
of
chemically
induced
stomatal
closure
requires
that
we
experiment
in
the
region
where
change
in
transpiration
per
change
in
path
length
S
is
greatest.
The
changes
in
transpiration
and
photosynthesis
per
change
in
S
are:
dT
D(Xf-Xp)
dP
D'(X'f-X'a)
dS
(L+
S)2
dS
(L+S+
M)2
The
diffusion
coefficients
and
concentrations
of
H20
and
C02,
in
the
surrounding
air,
will
be
unaffected
by
stomatal
closure.
1104
BOTANY:
ZELITCH
AND
WAGGONER
PROC.
N.
A
S.
If
the
concentration
of
water
vapor
X.
is
near
saturation,
(about
30
X
10-6
gm
cm-3
for
a
leaf
at
300)
and
the
stomata
are
open,
this
concentration
might
increase
to
37
X
10-6
when
the
stomata
close,
evaporation
decreases,
and
the
leaf
temperature
rises
to,
say,
34°.
The
effect
of
closure
upon
X'a,
on
the
other
hand,
cannot
be
clearly
predicted.
Thus
some
of
the
effect
of
closure
upon
transpiration
might
be
moderated
by
a
greater
gradient
(Xf
-
X,),
while
the
corresponding
effect
upon
photosynthesis
is
indefinite.
We
expect,
however,
that
the
effects
upon
the
diffusion
gradients
will
be
slight
compared
with
other
effects
that
we
will
now
discuss
under
the
assumption
that
the
numerators
D
(Xf
-
X,)
and D'
(X'f
-
X'a)
are
essentially
constant.
Under
environmental
circumstances
c,
the
transpiration
and
photosynthesis
are
labeled
T,
and
Pa.
In
that
neighborhood,
the
changes
in
these
two
quantities
caused
by
changes
in
stomatal
opening
can
be
simplified
by
substituting
from
equations
(1)
and
(2):
dT/dS=
-T/(L
+
S);
(5)
dP/dS
=
-Pe/(L
+
S
+
M).
(6)
Thus
a
change
in
both
quantities
T
and
P
will
be
obtained
most
dramatically
when
the
initial
levels
T,
and
Pc
are
high;
for
example.
when
plants
are
active
because
of
their
heredity
or
history,
or
are
exposed
to
a
high
light
intensity,
great
change
in
transpiration
or
photosynthesis
will
follow
induced
stomatal
closure.
The
effect
of
atmospheric
turbulence
is
less
obvious
intuitively,
but
is
clearly
shown
algebraically:
if
turbulence
is
great
and
L
is
therefore
small,
the
changes
in
loss
of
water
and
gain
of
CO2
will
be
greatest
for
a
given
change
in
stomatal
re-
sistance,
S.
Similarly,
the
changes
will
be
greatest
if
the
stomata
are
initially
wide
open
and
S
is
small.
All
of
these
factors
have
been
considered
in
the
design
of
the
experiments.
The
presence
of
M,
in
the
equations
for
photosynthesis,
suggests
that
the
fraction
of
photosynthesis
remaining
after
the
stomata
close
will
be
greater
than
the
fraction
of
transpiration
remaining.
This
can
be
seen
in
equations
(.5)
and
(6):
Al
reduces
the
change
in
P
below
that
in
T.
From
equations
(1)
and
(2),
one
can
show
that
the
ratios
of
the
processes
in
untreated
(subscript
0)
and
treated
(subscript
t)
leaves
are
(Pt1/Po)/(Tt1/To)
=
[(L
+
M
+
k5o)/(L
+
So)]
(L
+
St)/(L
+
Al
+
St).
(7)
This
index,
the
fraction
of
photosynthesis
remaining
after
treatment
relative
to
the
fraction
of
transpiration
remaining,
is
greatest
when
SO
is
zero
and
St
is
infinitely
large.
Thus
the
most
pronounced
effect
of
M
will
be
seen
if
we
compare
untreated
plants
whose
stomata
are
widest
and
treated
plants
whose
stomata
are
most
tightly
closed.
A
further
use
of
the
relative
values
of
T,/
TO
and
P1/PO
is
in
the
estimation
of
L
and
M.
The
regression
equation,
(4),
summarizes
all
observations
of
S
and
1/T,
or
S
and
1/P.
With
use
of
these
regressions,
first
T1/To
can
be
written
as
a
function
of
S,
which
is
calculated
from
observations
of
stomatal
width
by
means
of
equation
(3).
This
permits
a
calculation
of
L.
Similarly,
the
regression
of
1/P
on
S
permits
a
calculation
of
M.
All
of
these
predictions
are
valid
if
we
deal
with
the
simple
physical
system
specified
by
our
mathematics.
Since
transpiration
is
generally
conceded
to
be
VOL.
48,
1962
BOTANY:
ZELITCH
AND
WAGGONER
1105
understandable
in
the
rather
simple
physical
terms
of
potentials
and
resistances,
it
is
unlikely
that
we
have
gone
astray
in
our
specification
of
this
process.
Photo-
synthesis,
however,
involves
many
steps
beyond
the
supply
of
CO2
to
the
acceptor,
and
should
any
of
these
steps
be
hindered
by
the
treatment
designed
for
inducing
closure
of
stomata
alone,
photosynthesis
would
be
lower
than
our
prediction.
From
this
we
concluded
that
the
compound
which
closes
stomata
should
be
applied
as
directly
to
the
guard
cells
and
to
as
few
other
cells
as
possible.
Accord-
ingly,
the
spraying
technique
was
used.
We
also
concluded
that
a
compound
that
is
not
translocated
was
to
be
desired;
hence,
the
failure
of
phenylmercuric
acetate
to
move
from
upper
to
lower
guard
cells
(Tables
2
and
3)
was
an
advantage.
We
observed,
therefore,
the
changes
in
transpiration
and
photosynthesis
after
spraying
phenylmercuric
acetate
upon
tobacco
leaves.
The
untreated
leaf
had
open
stomata.
Photosynthesis
was
active,
turgidity
and
illumination
were
high,
and
ventilation
was
rapid
for
both
leaves.
If
the
photosynthesis
was
diminished
by
an
equal
or
smaller
amount
than
transpiration-,
the
compound
was
evidently
acting
on
little
else
but
guard
cells,
and
our
analysis
in
the
physical
terms
of
dif-
fusion
is
reasonable.
Methods.-On
Havana
Seed
tobacco
plants
growing
in
a
subirrigated
greenhouse
bench,
two
adjacent
leaves,
300
to
400
cm2
in
projected
area,
were
chosen
for
similarity
of
size.
One
leaf,
chosen
at
random,
was
sprayed
with
a
0.02%
Triton
X-100
solution,
and
the
other
was
sprayed
with
phenylmercuric
acetate
dissolved
in
the
Triton
solution.
The
pair
of
leaves
was
excised
from
1
hr
to
3
days
after
being
sprayed.
They
were
then
stood
in
small
beakers
of
water
upon
the
flooded
floor
of
a
transparent
chamber
(28
X
36
X
46
cm)
and
illumi-
nated
by
0.3
cal
cm-2
min'
for
1
hr,
conditions
which
were
found
to
insure
the
open-
ing
of
normal
stomata
as
determined
from
silicone
rubber
impressions.
The
leaves
were
sup-
ported
at
an
angle
of
490
above
the
horizontal.
Radiation
is
given
as
energy
below
3,000
mu
and
perpendicular
to
the
leaf.
The
radiation
came
from
water-cooled
incandescent
bulbs
60
cm
above
the
chamber
floor;6
they
delivered
two-thirds
as
many
quanta
per
calorie
as
does
the
sun.
With
no
turbulence
in
the
chamber
and
radiation
of
0.7
cal
cm-2
min-1,
a
mean
difference
of
2.2°
was
measured
between
a
30
gauge
copper-constantan
thermocouple
touching
the
upper
sur-
face
of
the
leaf
and
a
reference
junction
1
cm
above.
The
difference
was
reduced
to
about
0.30
by
spinning
a
22
cm
fan
inside
the
chamber
at
an
arbitrary
speed.
During
the
experiments,
the
fan
was
rotated
at
this
same
speed,
thereby
reducing
L
to
about
0.12
of
its
value
without
the
fan.
The
CO2
concentration
in
the
air
was
maintained
by
two
2.5
cm
diameter
openings
in
the
chamber.
The
air
within
the
chamber
was
kept
at
270
to
300
by
blowing
cool
air
on
the
thin
transparent
plastic
film
of
which
the
chamber
was
constructed.
This
maintained
the
dewpoint
at
about
220
by
condensation
of
water
inside
the
film.
At
time
zero,
which
was
one
hour
after
the
leaves
were
placed
in
the
chamber,
the
beakers
and
leaves
were
weighed,
radiation
was
increased
to
0.7
Cal
cm-2
min',
the
fan
was
turned
on,
and
the
measurements
of
transpiration
were
begun.
After
40
min,
the
two
openings
in
the
chamber
were
stoppered,
and
10
JAmoles
of
CO2
containing
4
,uc
of
C14
were
released
into
the
chamber.
This
increased
the
CO2
concentration
in
the
air
by
no
more
than
5
ppm.
After
exposure
of
both
leaves
to
C14
for
2
min,
the
ventilators
were
opened
and
fresh
air
was
swept
through
the
chamber.
At
the
expiration
of
60
min,
the
leaves
and
beakers
were
removed
and
weighed.
Stomatal
opening
was
determined
from
silicone
rubber
impressions
and
the
radioactivity
of
leaf
homogenates
was
measured.
The
observation
that
the
leaves
were
fully
turgid
both
at
the
beginning
and
end
of
the
experiment
was
confirmed
by
the
negligible
change
in
fresh
weight
of
the
leaves
and
in
the
stomatal
condition.
Results.
The
uniformity
of
the
leaves
and
of
the
environment
within
the
chamber
was
attested
by
the
behavior
of
untreated
leaves:
their
transpiration
ranged
only
from
6.1
to
11.4
mg
cm-2,
their
photosynthesis
from
1,140
to
1,950
counts
min'
1106
BOTANY:
ZELITCH
AND
WAGGONER
PROC.
N.
A.
S.
7g,
cm-2,
and
in
only
one
-g
~
of
18
experiments
were
00
-4
to
00
00
00
t0
00
t0
0
Lo
c
t-0
00
the
stomata
of
the
lower
O
-
4-
--
--
-
-
-
-_
-_
as
X
Zt
0
surface
of
control
leaves
X
0
,,
smaller
than
4.6,
in
0
°
;,
<,,
width
(Table
4).
The
°
*$;
-1~
CD
5;
Ct.
;J0
t-
U
X
S
CD
X.
ZC
f
Cleaves
identified
as
pairs
¢
33oo
g
O
O
-JC
t-
z
t~
0
t-
O
t-t~
u
z
dq
O
~1
to
9
were
taken
from
,
H
~0
the
same
lot
of
plants,
z
,,j
;.
<,
were
examined
on
nearly
-t
i-
o
~
consecutive
days,
and
co
N
V'.
M
t-n
M
t-
QC"t
M
-
t0
-4
O~
N
s
w
=
<;
000
x¢:,
-<5,
_,:i
AD,:i
<:i_
:>_ cr;
owere
exposed
to
aliquots
z
OCF
Q0n
of
the
same
radioactive
d
solution.
They
provide
=
> nt
c-o
o
~o
o
o
Qoo
xn
m
@z
auniformset.
oesi
1-
_
c_
0n
<
__
_
<
_
s
_
_
;The
phenylmercuric
acetate
sprayed
upon
the
z
~~~~0~
t
0tx
leaves
closed
the
stomata
¢
=;
-
-
-
-
n
n
n
8.O
of
these
plants
as
in
our
;~
E
E
E
fl
earlier
experiments,
pro-
z
Z:f
viding
stomatal
resis-
H
~~
~
~
n
n
g
Y
,,
tances
or
apparent
Cs
lengths
S,
which
were
;
^
d
o
_
N
n
e
<
s
S
x
=~3
estimated
from
the
meas-
m~
C
~
>
urements
of
stomatal
E-4
¢
.
>
2Wc
width
(Table
4)
bymeans
s.
=Xe
;0<>;
of
equation
(3).
The
S
E)
0°
°°
°
°° °°
°
°°
Lo
-t
t
1
,0t
Lo
0
ce
N
v
L,
ranged
from
O.05
to
0.21
t;
O
z-
____________.
°
-P
4T
cm.
The
clear
linear
re-
...4
a
T
j
lation
between
S
esti-
a2
-
E3
1g.
';=
.
mated
from
stomatal
*
.:"
s>
x:
x_.
>s
eX
'-
0;.
widths
and
the
recipro-
X
E-i
£
XO:,
Y
calsof
transpiration
(Fig.
¢
S'
1)
and
of
photosynthesis
Q4
<;
_>
ox
_
on9°~>t
@.
(Fig.
2)
verify
-anew
the
cc
'ct
dNc9
C.0
1-4
sc
00n
Nu
_1Y
Q
ot
_
=
t;
theory
introduced
to
C0-
botany
by
Brown
and
°-
Escombe
(equations
(1),
z
.
0
(2),
and
(4))
and
Penman
v
X
£;
and
Schofield's
relation
°
CM
o
oo
,
oo
o
between
aperture
and
S
f
a
(eq.
(3)).
The
statistical
w
1o
suitability
of
relating
00
oo
CC
oo
cm
O
m
co
co
oo
0
C
1/P
or
1/T
to
S
is
ap-
*,,
< <
parent
from
the
uniform-
0
;;;
<
ity
of
the
deviations
from
Cs
M
oC
d0
C.,
0
the
ca
lte
cre
A4
the
calculated
curves.
VOL.
48,
1962
BOTANY:
ZELITCH
AND
WAGGONER
1107
The
observations
from
different
plants
and
days
within
the
first
nine
pairs
show
the
surprising
uniformity
of
the
material
and
provide
an
explanation
of
the
varia-
bility
in
terms
of
stomatal
behavior.
.7
-
r2
86
.6
r2
.89
o
E
,
.5
20-
E
U|"
I-~~~~~~~~~~~a
o
15
1
5
lb
15
20
10
s,
cm
IOSS,
cm
FIG.
2.-The
1/P,
reciprocal
of
photo-
FIG.
1.-The
1/T,
reciprocal
of
tran-
synthesis,
as
a
function
of
S,
apparent
spiration,
as
a
function
of
S,
apparent
stomatal
length
(estimated
from
widths
stomatal
length,
for
leaf
pairs
1
to
9.
and
equation
(3)),
for
leaf
pairs
1
to
9.
An
index
of
the
fraction
of
photosynthesis
remaining
after
treatment
relative
to
the
fraction
of
transpiration
remaining
is
provided
by
equation
(7).
The
index
was
expected
to
be
greatest
when
the
change
in
S
is
large
and
the
remaining
transpiration
is
small.
Therefore,
we
selected
from
Table
4
those
experiments
where
the
remaining
transpiration,
i.e.,
transpiration
after
treatment,
was
no
more
than
three-quarters
of
the
control.
The
index
(equation
(7))
for
these
11
ex-
periments
ranged
from
1.40 to
0.96
with
a
median
of
1.07.
Since
this
index
was
greater
than
1.00
in
9
out
of
11
experiments,
we
conclude
that
the
fraction
of
photo-
synthesis
remaining
after
treatment
is
greater
than
the
fraction
of
transpiration
remaining.
(The
probability
of
observing
9
out
of
11
in
the
predicted
direction
by
chance
alone
is
0.03.)
The
regressions
(Figs.
1
and
2)
lead
to
estimates
of
L
and
M,
as
explained
earlier.
The
18
observations
of
S
and
1/T
correspond
to
L
=
0.07
cm.
This
estimate
and
the
18
observations
of
S
and
1/P
correspond
to
M
=
0.05
cm.
Thus
the
ratio
of
L:
S:
M
is
7:5:5
in
a
control
leaf
and
7:20:5
in
a
sprayed
leaf.
Others3'
4
have
observed
L
in
a
chamber
without
a
fan
to
be
1.3
to
1.6
cm.
The
change
in
temperature
gradients
in
our
chamber
showed
L
was
reduced
to
about
one-eighth
of
that
in
still
air
when
the
fan
was
operating.
Therefore,
the
estimate
L
=
0.07
is
reasonable.
As
stated
earlier,
our
measurements
of
the
index,
equation
(7),
and
estimate
of
M
will
be
reduced
to
the
extent
that
phenylmercuric
acetate
injures
biochemical
events
in
photosynthesis.
In
a
sense
M
indicates
injury
as
well
as
resistance
to
diffusion.
Since
the
median
of
our
observations
corresponds
to
values
of
M
lower
than
others
have
estimated,4'5
a
possibility
exists
that
compounds
more
specific
for
stomatal
closure
will
be
found
that
will
reduce
transpiration
with
even
less
disadvantage
to
CO2
assimilation.
Reducing
Transpiration
from
Intact
Plants.-The
preceding
theory
and
observa-
1108
CHEMISTRY:
CHAPMAN
ET
AL.
PROC.
N.
A.
S.
tions
on
excised
leaves
led
to
experiments
in
transpiration
control
in
whole
plants
grown
in
containers
in
the
greenhouse.
(Lack
of
space
prevents
our
reporting
here
the
experiments
in
which
a
foliar
spray
diminished
the
water
loss
significantly;
these
results
will
appear
in
the
August
issue
of
these
PROCEEDINGS.)
Summary.-The
standard
leaf
disk
assay
showed
that
at
concentrations
about
10-4
M
phenylmercuric
acetate
would
close
stomata.
The
effectiveness
revealed
by
the
disk
assay
has
been
correlated
with
the
results
of
foliar
spraying
and,
hence,
the
assay
is
advantageous
for
revealing
useful
compounds.
Phenylmercuric
acetate
sprayed
on
tobacco
or
maize
leaves
closed
stomata
only
on
the
leaf
surface
sprayed,
and
closure
persisted
for
at
least
14
days.
A
high
correlation
of
the
reciprocals
of
the
rates
of
transpiration
and
photo-
synthesis
with
resistance
of
diffusion
through
stomata,
which
was
calculated
from
stomatal
width,
was
predicted
from
diffusion
theory
and
has
been
verified
by
ob-
servation.
The
variation
in
stomatal
resistance
was
attained
by
spraying
phenyl-
mercuric
acetate
on
the
tobacco
leaves.
In
9
out
of
11
experiments,
induced
closure
of
stomata
reduced
transpiration
relatively
more
than
CO2
assimilation.
The
skillful
assistance
of
Isabelle
Namanworth
and
Dwight
Baldwin
Downs
is
gratefully
acknowledged.
*
This
investigation
was
supported
in
part
by
a
grant
from
the
National
Science
Foundation.
'Zelitch,
I.,
these
PROCEEDINGS,
47,
1423
(1961).
2
Blandy,
R.
V.,
Internatl.
Cong.
Crop
Protect.
Proc.,
4,
1513
(1957).
3Brown,
H.
T.,
and
Escombe,
F.,
Phil.
Trans.
Roy.
Soc.
London,
B193, 223
(1900).
4
Penman,
H.
L.,
and
Schofield,
R.
K.,
in
Symposia
of
the
Society
for
Experimental
Biology
(New
York:
Academic
Press,
1951),
vol.
5,
p.
115.
5
Gaastra,
P.,
Mededelingen
van
de
Landbouwhogeschool
te
Wageningen,
Nederland,
59,
1
(1959).
6
Rawlins,
S.
L.,
and
Moss,
D.
N.,
Agron.
J.,
54,
181
(1962).
THE
ISOLATION
OF
DEGRADATION
PRODUCTS
FROM
THE
ANTIBIOTIC,
ACTINOSPECTACIN
BY
D. D.
CHAPMAN,
R.
L.
AUTREY,
R.
H.
GOURLAY,
A.
L.
JOHNSON,
J.
SOUTO,
AND
D.
S.
TARBELL
DEPARTMENT
OF
CHEMISTRY,
UNIVERSITY
OF
ROCHESTER
Communicated
May
14,
1962
The
antibiotic,
actinospectacin"
2
has
recently3
been
shown
to
contain
a
1,3
bis(methylamino)tetrahydroxycyclohexane
unit
called
actinamine
(I).
Results
obtained
in
these
laboratories
are
in
agreement
with
the
proposed
structure,
and
in
this
paper
we
present
evidence
for
the
nature
of
the other
part
of
the
molecule.
Experimental.-The
N.M.R.
spectra
were
obtained
with
a
Varian
V-4300B
spectrometer
operating
at
60
Mc/s.
All
spectra
were
run
in
deuterochloroform
solution.
The
apparatus
and
method
described
by
Freeman4
were
used
for
the
decoupling
experiments.
A
Wilkens
Aerograph
gas
chromatographic
instrument
fitted
with
a
Wheelco
recorder
was
used
for
the
separation
of
the
liquid
products.
A
4'-
X
3/8q-
column
packed
with
10
per
cent
silicone
on
fluoropak
was
used.
... PheHg and MeHg also induce chromosomal aberration. Stomatal closure (Zelitch and Waggoner 1962) and PS II inhibition (Honeycutt and Krogmann 1972) are reported as PheHg phytotoxicity, too. These documented phytotoxic effects of PheHg and MeHg are partly similar to those of Hg(II), however, it remains elusive how plants detoxify the organomercurials. ...
... Re-examining the phenotypes of Arabidopsis plants exposed to PheHg previously reported (Bizily et al. 2003), we found similar segregation of root growth among seedlings, suggesting the specific toxicity of PheHg stress on the root development. Most of the studies on mercury phytotoxicity examine the effects of Hg(II) but a few studies reported PheHg-specific phytotoxicity (Zelitch and Waggoner 1962;Honeycutt and Krogmann 1972;Patra and Sharma 2000;Patra et al. 2004). Overall, the mutant phenotypes specific to PheHg stress suggest that possible conversion of PheHg to Hg(II) in the medium and/or plants is negligible under our experimental conditions. ...
Full-text available
Article
Key message An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Abstract Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.
... A smaller leaf area may influence the morphology, the functionally, and the number of stomata under drought conditions increasing the number of closed stomata and may reduce CO 2 absorption and limit biomass gain. The plant survival under drought conditions has promoted the use of tolerance parameters such as water-use efficiency (WUE) (Zelitch and Waggoner 1962). This index is based on the ratio between biomass production and water consumption (Kramer and Boyer 1995), and WUE is also calculated as the ratio between net photosynthesis and transpiration in gas exchange devices (Cruz et al. 2019). ...
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Article
This work aimed to evaluate the effects of lower water levels on leaf intercellular spaces and to assess their relations with the gas exchange, anatomy, and growth of Sorghum bicolor. Experiments were conducted in a greenhouse, in which plants were subjected to three water conditions (ten replicates, n = 30): well-irrigated, decreased irrigation, and limited irrigation. Lower water levels had no significant effect on the growth of S. bicolor but increased the biomass of the roots. Moreover, the number of leaves, leaf area, and leaf size as well as the chlorophyll content were not affected by lower water levels, and no significant changes were detected for whole plant photosynthesis, transpiration, or stomatal conductance. The water content of the plants and the water potential remained unchanged. However, compared with other treatments, the decreased irrigation decreased water loss and increased the water retention. Lower water levels increased the intercellular CO2 percentage, mesophyll area, and proportion of stomatal cavities and promoted minor changes in leaf tissue and stomatal traits. The increased stomatal cavities provided higher CO2 uptake and prevented excessive water loss. Thus, modifications to the intercellular spaces promoted conditions to avoid excessive water loss while concurrently improving CO2 uptake, which are important traits for drought-tolerant plants.
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The guard cells of Vicia faba and Nicotiana tabacum contain numerous mitochondria, elements of endoplasmic reticulum, spherosomes, and peroxisome-like microbodies. A full ribosomal complement appears in young but not in fully mature guard cells. Numerous small lipid droplets external to the plasmalemma were noted in mature Vicia guard cells. Chloroplasts were found in both epidermal and guard cells of both species. Full photosynthetic capacity was indicated by the grana fretwork of guard-cell chloroplasts. A specialized peripheral reticulum was observed in the guard-cell chloroplasts of Vicia. Plasmodesmata were observed in both walls between sister guard cells and between guard and epidermal cells. In the latter case plasmodesmata were found primarily in pit fields of transverse walls. It is postulated that the small volume of guard cells allows them an osmotic advantage over larger neighboring cells in generating turgor.