©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at
Phyton (Austria)
Special issue:
"P. J. C. Kuiper"
Vol. 40
Fasc. 3
(35)-(44)
31.3.2000
Red Light-Induced Acidification by Pea Leaf
Epidermal Cells is Regulated by More than one
Phytochrome
By
J. THEO M. ELZENGA 0 , MARTEN STAAL1} & HIDDE B.A. PRINS1}
Key
w o r d s : Phytochrome, signal transduction, acidification, cell expansion, Pisum
sativum.
Summary
ELZENGA J.T.M., STAAL M. & PRINS H.B.A. 2000. Red light-induced acidification by pea
leaf epidermal cells is regulated by more than one phytochrome. - Phyton (Horn, Austria) 40 (3):
(35) - (44).
Leaves of pea {Pisum sativum L.) grown in red light develop normally, provided that
functional phytochrome B is present. In the chromophore mutant pcd2 and in the phytochrome B
mutant lv the leaves remain small. Although some chlorophyll development takes place, the leaf
size of red light-grown mutant plant resembles that of plants grown in complete darkness. In white
light the leaf development in both mutants was comparable to wild type plants. The failure to
expand its leaves in the phytochrome mutants was not due to reduced cell division in the epidermis.
Leaves developed in red light had a similar number of cells as leaves grown in white light. The
difference in leaf size could completely be explaned by the difference in individual cell expansion.
Compared to the rate in wild type plants, the light-induced acidification, a major determinant of cell
expansion, was reduced in the phytochrome chromophore mutant and in the red light-grown
phytochrome B mutant, but was not reduced in white light-grown phytochrome B mutant. The role
of phytochrome in light-induced acidification and cell expansion is discussed.
Introduction
From seed germination to flowering, a limited set of pigment molecules
(either alone, in an antagonistic way or in a mutually dependent way) controls the
physiology of many stages of the life cycle of plants (BATSCHAUER 1998, CHORY
& al. 1996, REED & al. 1993, MOCKLER & al. 1999). A critical step in the
'' Department of Plant Biology, University of Groningen, P.O. Box 14, 9750 AA Haren,
The Netherlands.
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vegetative development is de-etiolation, a switch from typical dark growth
characteristics, like long and spindly stems, small folded leaves, no unfolding of
the apical hook and reduced pigmentation, to the normal light-grown
developmental pattern. The inhibition of expansion growth in the axial organs
(stems, petioles and hypocotyl) and the stimulation of expansion growth in the leaf
lamina, are two of the most rapidly initialized photomorphogenic effects in this
developmental process.
The control by light of expansion growth involves several light receptors
and affects multiple physiological processes. A key role is played by the protonpumping ATPase in the plasmamembrane of the leaf epidermal cells. Activation of
the ATPase lowers the pH of the cell wall compartment, resulting in activation of
expansion, which can break the hydrogen bonds of load bearing connections
between the cell wall components cellulose and hemicellulose (COSGROVE 1996).
Activation of expansin in this way increases the extensibility of the cell wall.
Activation of ATPase simultaneously provides the driving force for the uptake of
solutes, necessary for maintaining turgor pressure in expanding epidermal cells.
Light-stimulated extracellular acidification is fluence rate dependent, is
dependent on millimolar concentrations of potassium in the extracellular medium,
is blocked by barium, is inhibited by by the proton pumping ATPase inhibitor
DCCD and is insensitive to DCMU (STAAL & al. 1994). Both blue and red light act
as signals for activation of the proton pump (VAN VOLKENBURGH & al. 1990).
Most likely, phytochrome is the photoreceptor for the red light-induced
acidification. In dark-grown leaves the red light-induced acidification exhibits a
red/far-red reversibility (STAAL & al. 1994).
Of the phytochrome mutants that are isolated in Arabidopsis some have a
reduced cotyledon and leaf surface area, implicating a role for phytochrome in
expansion growth (REED & al. 1993, NEFF & CHORY 1998). The Argenteum mutant
of pea is deficient in a component of the cell wall middle lamella and as a result has
a very loosely attached leaf epidermal cell layer. Peeling this epidermal layer
affects the viability of the epidelmal cells minimally (HOCH & al. 1980), enabling
the study of the light response of this cell type without interference of the
mesophyll. In the present study phytochrome mutants of pea are used to study the
role of phytochrome in regulating proton pumping ATPase activity, cell growth
and tissue expansion in the leaf epidermis.
Materials
and
Methods
Seeds of the Argenteum mutant of pea (Pisum sativum) that lacks a component of the cell
wall middlelamella and enables the isolation of intact epidermal strips, were obtained from the Plant
Genetic Resources Unit, USDA/ARS, NYS Agricultural Experimental Station, Geneva, NY, USA.
The phytochrome mutants lv (WELLER & al. 1995), pcdl (WELLER & al. 1996), pcd2 (WELLER &
al. 1997a) and funl (WELLER & al. 1997b) were crossed with the Arg mutant. The original
phytochrome mutants (a kind gift from Dr. Weller, University of Tasmania, Hobard, Australia) were
backcrossed twice in the Arg background and the resulting plants were selved until an F4 was
obtained. Plants were grown in standard greenhouse mix soil either in white light, fluence rate 250
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^.s"1 provided by HPI-T 400W lamps (Philips, Eindhoven, The Netherlands) or in red light
with maximum wavelength at 660 nm and a half maximum bandwidth of 25 nm, 20 umol.nf2.s~'
provided by 36W/15 red fluorescent tubes (Philips, Eindhoven, The Netherlands) with a 12 h light/
12 dark regime at 20 °C.
The extracellular acidification was measured by placing a flat-tipped pH electrode (Ingold
Electrodes, Mettler Toledo, Tiel, The Netherlands) on the exposed inner surface of the epidermis,
removed from the abaxial surface of a leaf. Young leaves, still folded and approximately 40% of
full-size were used. Epidermal strips were incubated in a medium containing lmM KC1 and 5 uM
DCMU (to exclude possible interference of pH-changes due to photosynthesis by the guard cells).
In an earlier study it was shown that the pH changes thus observed, are due to the epidermal cells,
not due to the guard cells (ELZENGA & al. 1997). After mounting the epidermis and placing the
electrode the tissue was incubated in darknes for at least 45 min. The light from a slide projector
lamp (Capsule, 12V type 13083, Philips, Eindhoven, The Netherlands) was passed through a K65
(Balzers, Maarssen, The Netherlands) filter (central wavelength 650 nm). The fluence rate was
measured with a quantum meter (Quantiterm, Hansatech Instruments Ltd., King's Lynn, UK) at the
level of the epidermal strip. Changes in pH were continuously recorded on a chart recorder. After
termination of the experiment the viability of the epidermal cells was routinely checked by the
FDA/PI double staining method (OPARKA & READ 1994). Fluorescence of FDA and PI was
observed using a Nikon Diaphot inverted microscope (Nikon Inc. Tokyo, Japan) equipped with an
epifluorescence optic unit (filter combination Nikon B-2A: 510 nm dichroic mirror, 450-490 nm
excitation filter, 520 barrier filter). In all tissues examined more than 90% of the epidermis cells had
survived the isolation and the experimental procedure.
Before the start of the growth experiments plants were growing in either red or white light
for at least 10 days. The youngest leaflet that appeared separated from the plume was selected. With
two fresh razor blades, mounted in a holder 5.7 mm appart, a segment was cut from the middle of
the young unfolded leaf. With a single razor blade the central vein and the leaf edge were removed
and the remaining leaf segment was floated on a solution containing 10 mM KC1, 5 |iM DCMU and
0.5 % v/v EtOH. The segments were placed in contiuous 14 j4.mol.m"2.s"1 of red light (fluorescent
tubes, 36W/15 red, Philips) at 23 °C for 18 h. The strips were then measured, accurate to the
nearest 0.1 mm and then returned to the incubation medium and placed in 150 |amol.m~2.s"' white
light (fluorescent tubes TLD 18W/84, Philips) for 24 h. The capacity to grow in response to red
light was calculated from the percentage growth in red light:
100 x (LRED-5.7)/5.7 divided by the total growth defined as: 100 x (LRED +WHITE-5-7)/5.7.
Where, LRED is the length after the red light illumination and LRED +WHITE is the length after
red and white light illumination both in mm.
The surface area of epidermis cells was determined by mounting the epidermal strips
isolated from plants grown in either white or red light between two coverslips. A digital image was
either obtained by photographing the cells on Kodak gold 200ASA film for color prints and
digitizing the photos with a HP ScanJet Iicx, or by directly taking a digital image with a digital
camera (Coolpix 900, Nikon). The images were analyzed with the Sigma Scan Image measurement
software (Jandell Scientific, Erkrath, Germany).
DCCD,
N,N'-dicyclohexylcarbodiimide;
DCMU,
3-(3,4-dichlorophenyl)-l,ldimethylurea; FDA, fluoresceine diacetate; PI, propidium iodine
Results
To determine the role of phytochrome in the red light-induced extracellular
acidification by leaf epidermal cells and in light-induced expansion growth we
crossed the pcd2 and the Iv phytochrome mutants with the Arg mutant. The lv
mutant is defective in the apoprotein of phytochrome B. Other members of the
phytochrome family are not affected. Contrary, the pcd2 mutant is largly incapable
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of converting biliverdin IXa to 3Z-phytochromobilin (WELLER & al. 1997a), an
essential step in the biosynthesis of the phytochrome chromophore. As a
consequence this mutant has none at all, or very little phytochrome activity.
When grown in white light neither the lv, nor the pcd2 mutant has an
obvious phenotype. In red light the wild type plants still grow normally, but the
phy B mutant (lv) develops longer internodes while the leaves do no longer
expand, nor do they unfold (Fig. 1 and 2).
wild type
red light
pcd2
r
wild type
pcd2
white light
Fig. 1. Phenotype of wild type and lv (phy B) mutant of pea. After germination plants
were grown for 7 days either in red light (20 umol m"2 s"1) or in white light (100 pmol m"2 s"1). The
phy B plant grown in red light has less, but longer internodes, and very small, folded leaves.The phy
B plant grown in white light appears similar to the wild type plants. Phytochrome chromophore
mutants (pcdl and pcd2) behave identical to the lv mutant.
The tissue layer that is controling the leaf expansion growth rate is the
epidermis. In agreement herewith, the small leaf size of the red light grown phy B
mutant is mirrored in a reduction of cell expansion of the epidermal cells (Fig. 3),
showing that the small leaf size is due to reduced cell expansion, but that cell
division is unaffected. The lv (phyB) and pcd (chromophore) mutants do have
similar phenotypes.
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(39)
10 mm
I
10-
wild type
phy B
wild type
phyB
genotype
Fig. 2. Growth in red light of leaves of wild type and Iv mutant (phy B) plants. The apical
leaflets that were used for the measurements were still unfolded at the moment of measurement. The
lv leaves hardly grew after this stage.
To rule out the possibility that the growth reduction was due to a
permanent change (as opposed to a regulatory mechanism), we determined whether
the mutant leaves grown in red light, still have the potential to respond to other
light qualities. The growth rate of red-grown wild type and mutant plants was
determined in red light (Fig. 4.). As expected the growth rate of the mutant leaves
was lower than that of the wild type. From the growth response in white light we
can conclude that the capacity for light-stimulated growth is unaffected in the
mutants.
Reduction in expansion growth can be due to either, reduced acidification
of the cell wall compartment, or reduced capacity of the cell wall to respond to the
acidification (lower expansin concentration, heavier crosslinking of cell wall
polymers). In Fig. 5 a typical experiment is shown, in which the capacity to acidify
the extracellular medium in response to red light, of leaf epidermal strips of wild
type and phytochrome mutants is determined. The response to red light is reduced
in the phy B mutant. As an 'internal control' the response to blue light was also
determined and was found to be unaffected by the mutation. So, the capacity to
respond is intact and only the perception of the red light signal is impaired.
By comparing the red light-induced acidification with the response to blue
light it is also possible to cancel out the unintentional differences between
experiments (pH electrode placement, vitality of the cells in the epidermal strips,
etc.). Fig. 6 shows the relative response of red light-induced acidification of wild
type, chromophore (pcdl and pcd 2) and phy B mutants (lv) grown in either red or
white light.
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¥
>
phyB
white light
*
phyB
red light
50 M m
^ S r e d light
EnHlwhite light
w i l d type
wild type
white light _ ^ _ _ red light
50nm
a o.oo5(
J
a
I
0.002^
0.00OQ
phy B
white light
phy B
red light
phy B
wild type
Fig. 3. Effect of light quality and genotype on cell expansion (see Fig. 1 for experimental
details). The reduction in size of the epidermal cells explains the reduction in leaf expansion; the
number of epidermal cells/leaf is equal in all treatments.
Discussion
The development of leaves can strongly be influenced by environmental
conditions (reviewed by VAN VOLKENBURGH 1999). Especially light quantity and
quality and water supply modify growth characteristics. Light quality is sensed by
the plant through a limited set of sensory pigments, including phytochromes and
cryptochromes (ESKINS 1992, QiN & al. 1997). Arabidopsis cotelydons of plants
lacking phytochrome B fail to expand in red light (NEFF & VAN VOLKENBURGH
1994, NEFF & CHORY 1998) However, ROBSON & al. 1993 and CHORY 1992 report
stimulation of leaf expansion in phytochrome B mutants. Cotelydons of plants
lacking cryptochrome 1 do not expand in blue light (BLUM & al. 1994, LlN & al.
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1998). In addition to the stricktly photomorphogenic pigments, chlorophyll also is
capable of modulating the growth response of leaves to light (VAN VOLKENBURGH
&al. 1990).
The role of the epidermis in growth of organs is considered to restrict
expansion of underlying tissues (GREEN 1986, BECRAFT 1999). The driving force
for growth must be delivered by either the vascular tissue or the mesophyll. Since
in mature leaves the mesophyll cells are only loosely connected, become separated,
and form air spaces, the driving force probably is generated by the veins. In the
Argenteum mutant used in this study, the restrictive role of the epidermis is
demonstrated by the wrinkling of the leaves. This phenomenon is due to the loose
connection between mesophyll cells and epidermis cells, which allows the buckling
of the mesophyll layer within the two epidermal layers when the epidermal layer
stops expanding. Normal development of a leaf requires coordination of all tissues
involved (VAN VOLKENBURGH 1999). The observation that the small leaves of the
lv and pcd2 mutants developed in red light do not exhibit much wrinkling,
indicates that all tissues are reduced in growth.
T
25-
ESS red
I I red-white
•2015>105-
1
wildtype
phy B
genotype
pcdl
Fig. 4. The effect of light quality on the growth of leaf strips of wild type and mutant
plants. Strips were incubated in 1 mM KC1 and 5 \xM DCMU for 18 h in 14 (imol m'2 s"1 red light
and then measured (red), after that they were illuminate for 18 h in white light (150 jamol m"2 s"1)
and measured again (red-white). The growth in white light demonstrates that the mutants were not
affected in their ability to grow entirely.
The reduction in red light-induced growth of leaves of phytochrome
mutants of pea is due to a decrease of the cell expansion of the epidermal cells, not
to a decrease in cell division rate. This reduced cell expansion correlates with a
reduction of the red light-induced acidification of the cell wall compartment. In pea
epidermal cells the light-induced stimulation of cell expansion is thought to be the
result of increased proton pumping-ATPase activity (VAN VOLKENBURGH & al.
1990, STAAL & al. 1994, ELZENGA & al. 1997). For light-stimulated growth a
comparable mechanism was found (STAHLBERG & VAN VOLKENBURGH 1999). The
clear difference in acidification response to red light between the white light grown
phy B mutant and the chromophore mutants, could be due to a role for phy B in a
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developmental change that induces the epidermal cells to be responsive to red light,
enabling them to acidify the cell wall compartment and expand. Alternatively, the
red light induced acidification can be mediated by several phytochromes, one of
which is PHY B. Based on earlier experiments in which dark-grown wild type
plants still were capable of red light-stimulated acidification we favor this second
explanation. Preliminary data obtained with the funl mutant (phy A, data not
shown), indicate that the 'other' phytochrome is not PHY A.
• red on
0.0t
A
0.02-
red off
v blue on
A blue off
0.010.00-0.01 -
10
20
30
40
50
time (min)
Fig. 5. Red and blue light induced acidification by epidermal strips of wild type and phy B
mutant plants grown in red light. In the phy B mutant the response to red light is absent while wild
type plants still respond.
100i
"2 Is
I
I white growr
^ ^ 3 red grown
50-
wild type pcdl
pcd2
phy B
(chromophore mutants)
genotype
Fig. 6. Red light-induced acidification by wild type and mutant epidermal leaf strips
(expressed in arbitrary units). The phy B mutant (lv) does respond to red light when grown in white
light and not when grown in red. The chromophore mutants (pcdl and pcd 2) do not respond at all.
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