Botanical Studies (2008) 49: 235-241.
*
Corresponding author: E-mail: ffh@fct.unl.pt; Tel:
+351-21-2948500; Fax: +351-21-2954461.
INTRODUCTION
Many leaf chimeras arise from mutations that disrupt
chl synthesis or accumulation and are phenotypically
detectable by their lighter green color. It is well
established that most chl in chloroplasts serve only as
antenna pigments in photosynthesis, capturing incident
photons and transferring the resulting excitation energy to
reaction centers, where a minor, photochemically active
chl fraction uses it to drive a charge separation process.
Thus, mutations that cause only loss of chl, even if to a
relatively large extent, should not necessarily result in the
leaf ¡¦s inability to carry on photosynthesis. However, it has
been reported that pronounced chl deficiency affects the
synthesis and/or assembly of other chloroplast components
with which the chl interacts in vivo (Henriques and Park,
1975; Cumming and Bonnet, 1983; Green et al., 1988;
Klein et al., 1988). This is the case, for instance, of the
chl b-less mutant of barley, which is not only deprived of
chl b, but also of several major chloroplast polypeptides
that act as scaffolds for the assembly of the chl a, b light-
harvesting complex in the thylakoid membrane (Henriques
and Park, 1975; Burke et al., 1979). Over the past two
decades, it has been shown that a block in pigment
biosynthesis, either of chl or car, causes the accumulation
of intermediates that act as signals regulating the
expression of nuclear-encoded plastid-destined gene
products (Kropat et al., 1997; Rodermel, 2001; Strand et
al., 2003; Nott et al., 2006; Reinbothe et al., 2006). As a
consequence, leaf chimeras with largely reduced pigment
content often possess additional chloroplast alterations and
show more or less extensive photosynthetic impairments.
The functional characterization of these pigment
chimeras, thus, frequently proves to be rather complex,
and attempts to identify the specific change(s) responsible
for the photosynthetic disturbance(s) turn out immensely
challenging (Sommerville, 1986).
We report here data on pigment content and certain
photosynthetic characteristics of mutated leaves from
Photosynthetic characteristics of light-sensitive,
chlorophyll-deficient leaves from sectorially chimeric
stinging-nettle
Fernando S. HENRIQUES*
Plant Biology Unit, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
(Received August 8, 2007; Accepted March 12, 2008)
ABSTRACT.
Mutated leaves of sectorially chimeric stinging nettle (Urtica dioica L.) showed decreased
pigment content and reduced photosynthetic activity. Relative to their wild type siblings, the mutated leaves
were not only largely depleted in total chl and car, suggestive of a significant loss of photosynthetic units,
they also exhibited much higher chl a/b ra tios, indicative of a major reduc tion in the a ntenna size of the
remaining photosynthetic units. Light and electron microscopy confirmed a notable decrease in chloroplast
number in mutated leaves, and analysis of polypeptide composition revealed a large depletion of the
apoprotein of PSII antenna in these chloroplasts. The photosynthetic units present in mutated leaves showed
an intrinsic photochemical efficiency, measured as the variable to maximum fluorescence ratio of dark-adapted
leaves, only slightly lower than controls, and their concentration correlated strongly with the leaves¡¦s net
CO
2
uptake capacity. It is concluded that mutated leaves of stinging nettle underwent a gradual chloroplast
loss during development, but the remaining organelles preserved much of their photosynthetic competence.
This chloroplast loss most likely arises from an accumulation of chl precursors that repress the synthesis of
photosynthetic essential proteins and act as photosensitizers for chloroplast degradation.
Keywords: Chimerism; Chlorophyll fluorescence; Chloroplast peptides; Photosynthetic net CO
2
uptake;
Urtica dioica L.
Abbreviations: Car, Carotenoids; Chl, Chlorophyll; F
O
, F
m
, F
v
, Minimum, maximum and variable
fluorescence of dark-adapted leaves, respectively; LHC-II, Light-harvesting chl a,b-protein complex of
PSII; PQ, Plastoquinone; PSI, PSII, Photosystems I and II, respectively; PSU, Photosynthetic unit; Q
A
, PSII
primary electron acceptor.
PHYSIOLOGY
pg_0002
236
Botanical Studies, Vol. 49, 2008
sectorially chimeric stinging-nettle at two defined
developmental stages. Relative to controls, mutated
leaves are largely depleted in both chl and car and possess
fewer chloroplasts; the photosynthetic units (PSUs) of
these remaining chloroplasts are of a smaller size due to
extensive loss of their antennae subunits, but are only
slightly disabled in their photochemical competence, and
their concentration correlates highly with the leaf¡¦s net
CO
2
uptake capacity. It is proposed that the smaller size
of the PSUs present in the mutated leaves results from a
block in chl biosynthesis that represses the expression of
the light-harvesting chl a, b apoprotein genes. It is further
proposed that the build up of chl precursors in mutated
leaves impairs the normal turnover of proteins that makeup
the photosynthetic apparatus and causes photooxidative
damage to the thylakoid membranes, ultimately accounting
for the chloroplast loss.
MATERIALS AND METHODS
Plant material and growth conditions
This study was carried out with normal and sectorially
chimeric stinging-nettle (Urtica dioica L.) plants grown
in open air, under natural sunlight. The chimeric plant
initially presented a typical, full green appearance, but
it later developed a mutated lateral shoot of a uniformly
lighter green color. All axillary buds originating from
this shoot propagated the mutation, and the adult plant
thus possessed branches with normal (control) leaves and
branches with mutated leaves. Plants were watered and
fertilized regularly, and measurements were carried out on
leaves tagged on appearance and followed throughout their
developmental course.
Chloroplast isolation and pigment determination
Three grams of deveined normal leaves (or 10 g of
chimeric leaves) kept in liquid nitrogen were finely
broken with a mortar, 25 ml of sucrose-phosphate buffer
[0.4 M sucrose, 0.15 M KCl, 2% sodium ascorbate,
1% polyvinylpyrrolidone, and 0.05 M potassium
phosphate buffer (pH 6.8)] were added, and the slurry
was homogenized by 2¡Ñ5-second bursts in a Waring
blender. The homogenate was strained through 8 layers
of cheesecloth and centrifuged at 1,000 xg for 10 min
to pellet down the chloroplasts (Henriques, 2004). Chl
and car were extracted with 80% acetone and quantified
spectrophotometrically using the equations of Porra et
al. (Porra et al., 1984) and Lichtenthaler (Lichtenthaler,
1987), respectively.
Gas exchange and chlorophyll fluorescence
measurements
Measurements of leaf net CO
2
uptake were performed
with an open, flow-through gas exchange system (LCi,
Hansatech, King¡¦s Lynn, Norfolk, England) at ambient
CO
2
levels, as described before (Henriques, 2003). In vivo
chlorophyll fluorescence parameters were recorded using
a direct portable fluorometer (PEA, Hansatech Ltd, King¡¦s
Lynn, Norfolk, England). Leaves were dark-adapted for
30 min before measurements were taken. Initial (F
0
) ,
maximum (F
m
) and variable (F
v
= F
m
-F
0
) fluorescence
yields as well as the F
v
/F
m
ratio were recorded. The area
over the fluorescence induction curve measured in DCMU
treated leaves (Srivastava et al., 1997) was also recorded.
This area is directly related to the number of Q
A
molecules
and is taken to indicate the number of active PSII units per
illuminated leaf area volume, hereafter referred to as PSII
concentration. Given the small differences in F
v
/F
m
found
between the control and mutated leaves, this approach
was used to compare PSII concentrations between the two
genotypes.
Protein preparation and SDS-polyacrylamide
gel electrophoresis
After isolation, chloroplasts were twice washed with
a 0.05 M phosphate buffer (pH 7.4) containing 0.15 M
KCl, incubated for 30 min in 1 mM EDTA (pH 8.0),
and centrifuged at 15,000 xg for 10 min. EDTA-washed
chloroplasts were lipid-extracted several times with a
chloroform:methanol mixture (1:2, v:v), followed by
3 washes with anhydrous methanol, and the resulting
protein was dried under vacuum (Henriques and Park,
1975). The dried protein was dissolved in 0.0625 M Tris-
HCl (pH 6.8), 5% glycerol, 5%
£]
-mercaptoethanol and
2% SDS at a concentration of 1 mg protein/ml, incubated
overnight at 37oC and heated in boiling water for 2
min. For electrophoresis, a 1.5 cm long, 3% acrylamide
stacking gel (pH 6.8) and a 9.0 cm long, 9% acrylamide
separating gel were used. The discontinuous SDS-PAGE
system of Laemmli was used (Laemmli, 1970). Gels were
stained with Coomassie Brilliant Blue R overnight and
destained in methanol:water:acetic acid (50:875:75, v:v:v)
as described before (Henriques and Park, 1975). Molecular
mass estimates were determined using Bioscience low-
range protein standards (Amersham, Malmo, Sweden).
Microscopy studies
Leaf segments were fixed in chilled 2%
glutardialdehyde in 50 mM Na-phosphate buffer (pH 7.4)
and post-fixed in 1% osmium tetroxide and dehydrated in
a graded series of ethanol solutions. For light microscopy,
the slices were embedded in methyl methacrylate and
sectioned; thick sections were mounted on clear glass
slides, stained with 1% toluidine blue and observed
with a Leitz optical microscope. For scanning electron
microscopy, the leaf slices were dried to the critical
point, mounted on a specimen stub, coated with gold,
and examined in a Jeol JSM 35 CF scanning electron
microscope.
Data presentation and statistical analyses
Data presented are the mean¡ÓSD of three independent
experiments each with three to five replications.
Comparisons between means were carried out by one-way
pg_0003
HENRIQUES ¡X Photosynthesis in chimeric stinging-nettle
237
ANOVA (F-ratio test). Different letters indicate significant
differences at P
.
0.05.
RESULTS
The sectorially chimeric stinging-nettle plant here
described developed axillary branches with mutated leaves
of identical genotype and uniform color. The mutated
leaves were initially of a light-green color (hereafter
referred to as young expanded leaves), turning to a yellow-
greenish color after full expansion (hereafter referred to
as mature expanded leaves) and whitening subsequently.
Mean pigment contents and net CO
2
uptake of mutated and
control leaves are presented in Table 1. Data on pigments
show that during the development of mutated leaves,
the contents of both chl and car decrease progressively,
reaching ca. 20% and 25%, respectively, of control values
in mature expanded leaves. This loss of total pigment
is accompanied by a significant increase in chla/b and
decrease in chl/car ratios, indicating that there occurs a
preferential loss of chl b and a relative enrichment in car
during the ontogeny of mutated leaves. These changes in
pigment levels and ratios reflect major alterations in the
number and composition of PSUs in mutated leaves, as
will be discussed below.
Table 1 also compares net photosynthetic rates of
control and mutated leaves. The net CO
2
uptake rate
of control leaves is within the range reported for many
herbaceous C
3
species and is significantly higher than those
of mutated leaves on an area basis. Photosynthetic rates of
young and mature expanded mutated leaves decreased to
ca. 64 and 24% of control, respectively. The extent of this
reduction in the photosynthetic rates of mutated leaves is
significantly smaller than the extent of their losses in chl
content, thus indicating that some of the lost chl served
only as antenna pigment and was not essential for CO
2
assimilation in the conditions under which measurements
were carried out. This becomes immediately apparent
when photosynthetic rates are expressed on a chl weight
basis, which reveals that mutated leaves display higher
photosynthetic rates than the controls (Table 1).
Control leaves showed an F
v
/F
m
ratio of 0.824
¡Ó
0,006
(Table 2), identical to those reported previously for
leaves of higher plants (Bjorkman and Demming, 1987).
This F
v
/F
m
ratio underwent a small, though statistically
significant, decrease in young expanded mutated leaves
and fell slightly further in older mutated leaves. Identical
F
v
/ F
m
values were found when the measurements were
carried out at pre-dawn and after a 30-min dark adaptation.
Mutated leaves also showed rather extensive decreases in
their PSU concentrations, amounting to about 30 and 70%
for young and mature expanded leaves, respectively (Table
2). The concentration of the remaining PSUs was found
to correlate strongly with the measured photosynthetic
rates (y=0.0523x+0.031, R
2
=0.9832), thus confirming the
PSUs present in mutated leaves retain much of their full
functional competence.
Figure 1 shows that the polypeptide composition
of chloroplasts from normal and mutated leaves is
qualitatively identical although some gross quantitative
differences are immediately apparent, particularly in the
55 and 27 kDa regions. Most conspicuous in chloroplasts
from mutated leaves is the large depletion of a protein
band in the 27 kDa region of the gel, but the smaller
peak running ahead of the 27 kDa band also showed a
large decrease. The peak at 55 kDa corresponds to the
large subunit of Rubisco (Henriques and Park, 1976),
and its increase in the mutant profile results mostly from
the relative decrease in the 27 kDa component for an
equivalent amount of protein loaded onto the gels, but
also from some unstacking that occurs in the chloroplasts
of mutated leaves and that provides additional area
for Rubisco adsorption. Note that the peak at 15 kDa,
corresponding to the small subunit of Rubisco, is likewise
enriched in the polypeptide profile of chloroplasts from
mutated leaves.
Figure 2 shows scanning electron micrographs of
normal (A) and expanded mutated (B) leaves from
stinging-nettle. The transverse sections of mutated leaves
showed no obvious morphological alteration relative to
controls, exhibiting a characteristic mesophyll arrangement
Table 1. Pigment content and photosynthetic net CO
2
uptake
rate of control (mature leaf) and mutated leaves of chimeric
stinging-nettle.
Variable
Treatment
Control Young
mutated
Mature
mutated
Total chl (
£g
g¡Pcm
-2
)
33.0
¡Ó
1
a
13.4
¡Ó
1
b
6.8
¡Ó
1
c
Chl a/b
3.3
a
4.6
b
7.5
c
Total car (
£g
g¡Pcm
-2
)
8.0
¡Ó
0.5
a
3.7
¡Ó
0.5
b
2.1
¡Ó
0.5
c
Chl/car (molar ratio)
2.6
a
2.1
b
1.9
c
Net CO
2
uptake (
£g
mol¡Pm
-2
¡Ps
-1
) 17.6
¡Ó
1
a
13.4
¡Ó
1
b
4.2
¡Ó
1
c
Net CO
2
uptake (
£g
mol¡Pmg
-1
chl¡Ps
-1
) 0.053 0.1 0.062
*
Results are the mean of three values
¡Ó
SD of three
independent experiments. Different letters indicate
statistically significant differences at P
.
0.05.
Table 2. Variable to m aximum chl fluores cence ra tio and
concentration of PSII units in control (mature leaf) and mutated
stinging nettle leaves.*
Control Young mutated Mature mutated
F
v
/F
m
0.824
¡Ó
0.006
a
0.799
¡Ó
0,008
b
0.766
¡Ó
0.009
c
PSU conc. 100%
68%
27%
*
Results are the mean
¡Ó
SD of at leas t ten m easureme nts .
Different letters mean statistically significant differences at P
.
0.05.
pg_0004
238
Botanical Studies, Vol. 49, 2008
with an upper palisade and a lower spongy parenchyma.
A large hair, with a multicellular basis, and a smaller one
were visible in the lower epidermis of control and mutated
leaves, respectively; stomata were restricted to the lower
epidermis. Control cells that were fractured during leaf
sectioning revealed the presence of a large number of
chloroplasts (Figure 2C); however, far fewer chloroplasts
were observed in mutated leaves, where cells were often
collapsed. In an attempt to place this difference into a
more quantitative perspective, leaf sections of control and
mutated leaves were also examined by light microscopy.
Figure 3 shows that control cells (Figure 3A) possessed
a large number of chloroplasts arranged in a compact
manner in the cell periphery while young expanded
(Figure 3B) and mature expanded (Figure 3C) mutated
leaves contained progressively fewer organelles. Given
some variability among examined leaf samples, it can be
only concluded that mature expanded leaves contained
somewhere between one-third and one-fourth as many
chloroplasts as controls.
DISCUSSION
The results presented show that, on an area basis,
the mutated leaves of chimeric stinging-nettle have a
decreased photosynthetic capacity compared to their
wild type siblings and that the magnitude of this decrease
augments from young expanded to mature expanded
leaves. It is further shown that this photosynthetic decline
is highly correlated with the loss of PSUs per unit leaf
area, but only indirectly related to the reduction in leaf chl
content. This indicates that part of the lost chl served only
as antenna pigment and is consistent with the much higher
chl a/b ratios observed in mutated leaves, which reveal a
preferential loss of chl b relative to chl a. In PSII, chl b is
exclusively located in the LHC-II antenna [note that the
term LHC-II is used here to refer to the whole chl-a, b
light-harvesting chl-protein complex associated with PSII,
comprising both the minor, tightly-bound LH-CIIa (CP29),
LH-CIIc (CP26), LH-CIId (CP24), and PsbS (CP22)
complexes, as well as the major, reversibly-bound LHC-IIb
containing the Lhcb1/2 gene products], a family of closely
related, nuclear-encoded chl a, b-binding polypeptides
organized around the PSII core (Jansson, 1994; Green
and Durnford, 1996). Recently, high resolution structural
studies of the Lhcb1 and Lhcb2 subunits of the complex
revealed the presence of 14 chls per polypeptide, 8 chls
a and 6 chls b, as well as 4 carotenoids (Liu et al., 2004).
There are no such detailed studies for most of the other
LHC-II subunits, but biochemical analyses suggest that,
Figure 2. Scanning electron micrographs of cross-sections from normal (A) and mutated (B) leaves. C shows a magnified view of a
ruptured control mesophyll cell with several chloroplasts.
Figure 1. Polypeptide profiles of EDTA-washed, lipid-extracted
chloroplast membranes from normal (B) and mutated (C) leaves
of stinging-nettle; approximate equal amounts of total protein
were loaded in the two lanes. Lane A shows the mol wt markers.
Arrows on the right indicate major differences found between
the mutant and wild type.
pg_0005
HENRIQUES ¡X Photosynthesis in chimeric stinging-nettle
239
in spite of some variability in their chl/protein ratios, their
chl a/b ratios are not markedly different from that of the
Lhcb1/2 monomers (Jansson, 1994; Green and Durnford,
1996), which would yield an overall chl a/b ratio of near
1.5 for the bulk LHC-II complex against an overall 3.3 for
whole chloroplasts of stinging-nettle. Thus, removal of the
chl b-enriched LHC-II subunits would decrease chl content
and raise chloroplast chl a/b ratios, but should have no
direct negative effect on PSII photochemical activity,
as was found here. In fact, the F
v
/ F
m
ratio in younger
mutated leaves is rather close to that of controls, and this
ratio decreases only slightly in older mutated leaves. The
F
v
/F
m
ratio of dark-adapted leaves estimates the intrinsic
quantum efficiency of PSII photochemistry, and the values
measured in mutated leaves indicate that the remaining
PSII units have undergone only minor impairments
in their photochemical capacity. We cannot ascertain
unequivocally the causes for this F
v
/F
m
ratio decrease
in mutated leaves, but the fact that it is also observed
at pre-dawn strongly indicates that it arises from the
presence of a number of damaged PSII units concurrent
with normal, fully-functioning ones. These damaged
photosynthetic units absorb light energy but are unable to
perform photochemistry, and thus lower the F
v
/F
m
ratio
of the mutated leaves. In any case, it is clear that the PSII
units remaining in the chloroplasts of mutated leaves
retain most of their photochemical capacity, in spite of an
extensive loss of their antenna complex. This conclusion
is supported by comparative analysis of chloroplast
polypeptide profiles from normal and mutated cells that
revealed major differences between the two in the 25 to
27-kDa region. It is known that the polypeptides in this
range comprise the protein moiety of the LHC-II complex
(Henriques and Park, 1975; Burke et al., 1979), and their
depletion in the chloroplasts from mutated cells further
attests to the loss of a significant part of this complex. In
particular, the major LHCII-b component appears to be
largely missing. This PSII antenna component is relatively
depleted in carotenoids, displaying chl/car ratios of 4.0 to
5.5 (Lichtenthaler et al., 1981; Lichtenthaler and Babani,
2004), and its preferential loss is revealed by the lower
cha/b ratio found in mutated leaves.
The results just discussed suggest two immediate
alternative explanations for the losses in the PSII-
antenna components observed in mutated leaves. The
first is that the primary effect of the mutation was to
partially block chl biosynthesis, causing an accumulation
of its precursors that, in turn, negatively controlled the
expression of the apoprotein of LHC-II. The second is
that the mutation originally affected the synthesis of the
LHC-II polypeptides, thereby resulting in the absence of
the protein scaffold to which the pigments would have
anchored, which turned off their biosynthesis. One cannot
select between these two alternatives based on the present
experimental data but numerous genetic and biochemical
studies, namely with a number of chl-deficient mutants
(Cumming and Bonnet, 1983; Green et al., 1988; Klein
et al., 1988), have shown that chl is necessary for both
the synthesis and proper assembly of the LHC-II, thus
favoring the first alternative. Recently, Reinbothe et
al. (2006) showed that a lack of capacity to convert
chlorophyllide a into chlorophyllide b in the chloroplast
inhibits the import and stabilization of chl-binding light-
harvesting proteins, further supporting the first alternative.
Future work will provide a more solid basis from which to
judge between the two alternatives.
Acknowledgements. The author thanks the excellent
technical assistance of Ms. Isabel M. Portant.
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HENRIQUES ¡X Photosynthesis in chimeric stinging-nettle
241