Plant Physiol. (1985) 77, 29-34
0032-0889/85/77/0029/06/$01.00/0
Light Quality Effects on Corn Chloroplast Development
Received for publication July 23, 1984 and in revised form September 19, 1984
KENNETH ESKINS*, MURRAY DUYSEN, LINDA DYBAS,
AND
SUSAN MCCARTHY
Northern Regional Research Center, Agricultural Research Service, United States Department of
Agriculture, Peoria, Illinois 61604 (K.E., S.M.); Botany Department, North Dakota State University,
Fargo, North Dakota 58105 (M.D.); and Botany Department, Knox College, Galesburg, Illinois 61401
(L.D.)
small amount of R, no differences between the mutant and
normal genotypes are expressed, but both types have high ratios
of PSII/PSI complexes and unusual grana development (10).
We have now examined another cell type differentiation process and the effects of various light qualities on its expression.
We chose corn mesophyll and bundle sheath cells because they
provide an opportunity not only to investigate the effect of light
quality on development of the chloroplast, but also its effects on
the cooperative development of cell types that are interdependent. In this first report, however, only young tissue is used and
the effects of light quality are the primary interest.
We have chosen a broad range of light conditions to examine
these effects. Sweet corn was grown in a greenhouse under
supplementary light at 50 w/m2 in R at 11 w/m2, and in a FR
source that contains a small amount of R at 9 w/m2. Mesophyll
and bundle sheath cells were isolated and examined for pigment
content, membrane polypeptides, pigment-protein complexes,
and ultrastructure.
ABSTRACT
Corn was grown under greenhouse and controlled light quality conditions incluing full spectrum, red (R), and far-red (FR) sources. Young
leaf samples were analyzed for pigments, pigment-proteins, membrane
polypeptides, and ultrastructure. Chloroplast development in full spectrum white light was similar to that found in R but different from that
found in FR plus low R. Compared to greenhouse and R, FR plus low R
(670-760) repressed the formation of photosystem I reaction center
protein (CPI + CPla) and enhanced those of photosystem II (CPa) in
both bundle sheath and mesophyll cells. Photosystem II polypeptides
were present in both cell types, with the 46 and 34 kilodalton proteins
predominant in mesophyll cells. Bundle sheath cells contained relatively
more of the 51 kilodalton and less of the 46 kilodalton proteins. However,
they also contained measurable amounts of ribulose bisphosphate carboxylase which may interfere with estimates of the 51 kilodalton protein.
MATERIALS AND METHODS
Growth Conditions. Corn (Zea mays OP Golden Bantum) was
planted in plastic trays (36 x 30 x 15 cm) in a mixture of
vermiculite and top soil 3.1 (v/v), well watered, and covered with
black plastic until germination occurred (2-3 d). The trays were
then placed in the greenhouse under supplemental fluorescent
and incandescent lights (total fluence rate of 50 w/m2), 14 h light
and 10 h dark, or under special fluorescent lights in a growth
chamber described below, also for 14 h light and 10 h dark
samples were picked from the first emergent leaf, usually 9 to 10
d after planting.
Light Sources. Greenhouse conditions were supplemented by
eight cool-white fluorescent tubes (8 ft, 1500 mamp VHO)
supplied by GTE Sylvania and by eight 50-w incandescent bulbs.
R at 11 w/m2 was supplied by Red-GTE Sylvania type 236
emission peak at 660 nm, halfbandwidth 18 nm. The extraneous
blue and yellow bands of these lights were removed by a plastic
filter, Roscolux no. 19 'Fire', 0% transmission at 550 nm. FR at
9 w/m2 was obtained by filtering Far-red GTE Sylvania type 232
emission peak at 740 nm, half bandwidth 80 nm with Roscolux
no. 19 'Fire' (emission spectra, Fig. 1).
Pigment Analysis. Chloroplast pigment analysis was by means
of an HPLC analysis procedure using reverse phase chromatography (9).
Chloroplast Isolation. Ten d after planting, the newly emergent
leaf was removed, placed on ice, and torn into thin strips (2
mm). These strips were further cut into small sections with
scissors, then placed in an ice cold Waring Blendor in 100 ml of
ice cold buffer [0.3 mM sucrose, 10 mM KCI, 50 mM K2HPO4
(pH 8.0)], and blended at 50% offull power for 4 s. The resulting
blend was filtered through a milk filter and two layers of Miracloth (20 ,um). The filtrate was centrifuged at 2000g for 5 min.
The effects of light quality on higher plant chloroplast development is being examined with increasing frequency and enthusiasm (1, 19, 23). Although it has been known for some time
that light intensity and light quality affect parameters of chloroplast development such as the type of grana, the relative amounts
of pigment-protein complexes, membrane polypeptides, and
electron transport constituents (5, 6), specific information on the
interaction of light quality and cell type is lacking. Also, recent
evidence that gene expression may be under light control has
opened up exciting new possibilities for managing plant development (4, 18).
In line with this aspect of light quality research, our recent
work has concentrated on the effect of light intensity and light
quality on the expression of genetically determined pigment
deficiency in soybeans (10). In high light intensity (field conditions) the mutant soybean genotypes, when compared to the wild
type, exhibit delayed development and deficiency of the lightharvesting complex. In addition, they also show a reduced ratio
of the PSI reaction centers relative to PSII centers (8). Development of the light-harvesting complex is under the direction of
nuclear genes (15), whereas the synthesis of reaction center
complexes is under the control of chloroplast genes (21). Under
medium light intensity (18 w/m2) of either full spectrum white
light or blue light, only the nuclear gene-controlled light-harvesting complex deficiency is expressed (7). Under FR' containing a
'Abbreviations: FR, far-red light; R, red light, CPa, pigment-protein
complex associated with PSII; CPI, pigment-protein complex associated
with PSI; FP, free pigment; LHC, light-harvesting complex; LHPP, lightharvesting pigment protein; TLH, total light harvesting; RuBPCO, ribulose bisphosphate carboxylase.
29
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30
Plant Physiol. Vol. 77, 1985
ESKINS ET AL.
Table I. Pigment Content of Corn Leaf in Various Lights
Pigment Content
Pigment
Greenhouse
R
FR + low R
(50 w/m2)
(11 w/m2)
(9 W/m2)
nmol/gfresh wt
Neoxanthin
59
52
23
Violaxanthin
95
71
39
71
192
Lutein
213
174
144
64
Carotene
505
Chl b
476
207
817
Chl a
1670
1840
E
E
3'
1.0
650
Wavelength, nm
FIG. 1. Emission spectra of light sources: R (GTE Sylvania type 236)
and FR + low R (GTE Sylvania type 232). Measured by power meter
(United Detector Technology, Inc., model 40X) through monochromator (6.4 nm bandwidth) at 20 cm from source. Lights filtered by 10 cm
H20 and one layer of Roscolux No. 19 'Fire' plastic filter (Rosco Co.,
Port Chester, NY). Both the monochromator and the power meter
showed flat response from 550 to 800 nm.
The resultant pellet was washed with ice cold water, recentrifuged
at 4000g, and taken up in 1 mm EDTA (pH 8.0). This was
pelleted (l0,000g) and then washed in 50 mM Tricine (pH 8.0).
After centrifugation at 10,000g, the pellet from this wash (mesophyll fraction) was taken up in isolation media and stored at
liquid nitrogen temperatures until further use. The residue from
the original filtration was washed well with isolation media and
then blended at full power for 30 s. The blend was then filtered
as described above. The filtrate was discarded and the procedure
repeated on the residue. The second filtrate was also discarded,
and the residue was then blended at full power for 1 min. This
blend was again filtered through a milk filter and two layers of
Miracloth (20 gm), then centrifuged at 4000g for 5 min. After
washing the pellet with ice cold water, the suspension was treated
as described above for the mesophyll fraction; then the pellet
(bundle sheath fraction) was taken up in isolation media and
stored in liquid nitrogen.
Gel Electrophoresis. Pigment-proteins complexes and membrane polypeptides were analyzed by modified procedures of
Anderson et al. (2) and of Guikema and Sherman (13), respectively. Experimental conditions were as previously described (10).
Electron Microscopy. Samples were taken from the middle of
the young leaf at a point between the midrib and the leaf edge.
Samples were fixed in 2.5% glutaraldehyde in 0.1 M Millonig's
buffer overnight and then, after washing with Millonig's buffer,
were further fixed in 2% Os04 in Millonig's buffer for 3 h. The
fixed samples were dehydrated by a series of graded alcohol
washes, then embedded in Spurr low-viscosity medium and cured
overnight at 80°C. The embedded samples were cut with a glass
knife, stained, and viewed with a Hitachi HS-75 electron microscope.
RESULTS
At least two principal questions may be addressed by our
current data: how do mesophyll and bundle sheath cells differ,
and how do they respond to light quality stimuli? First, we
determined the major differences in pigments, pigment-protein
complexes, membrane polypeptides, and ultrastructure between
young corn mesophyll and bundle sheath chloroplasts. In this
regard, special emphasis has been placed on the proteins associated with the reaction centers of PSI and PSII and the lightharvesting complex because there are major differences in their
Ratio of Chl a/Pigment
28.3
35.8
17.8
26
7.8
9.6
9.5
12.8
3.3
3.8
Neoxanthin
Violaxanthin
Lutein
Carotene
Chl b
36.0
20.8
11.4
12.7
3.9
Table II. Molar Ratios of Chl a/Accessory Pigments for Corn
Mesophyll and Bundle Sheath Cells
Chl a/Accessory Pigments
Pigment
Greenhouse
(50 W/m2)
R
(11
w/m2)
FR + low R
(9 w/m2)
molar ratio
Mesophyll
Neoxanthin
Violaxanthin
Lutein
Carotene
Chlb
Bundle sheath
Neoxanthin
Violaxanthin
Lutein
Carotene
Chlb
25.0 ± 0.8a
19.2 ± 0.2
8.4 ± 0.6
10.0± 0.2
3.4±0.2
26.5 ± 0.4
22.0 ± 0.4
8.9 ± 0.2
10.6 ±0.1
3.4±0.1
22.1 ± 1.9
15.4 ± I
8.5 ± 0.9
9.8 ±0.1
3.2±0.1
46.0 ± 0.2
23.6 ± 0.4
12.7 ±0.1
12.3 ± 0.3
4.9 ±0.4
42.8 ± 0.6
22.0 ± 1.0
10.4± 1.1
12.0 ± 0.5
4.5 ±0.3
43.8 ± 22.5
15.2 ± 0.8
9.1 ± 1.8
10.0 ± 0.5
4.8 ±0.2
'Mean ± SD.
composition in the two cell types. Second, we examined how
light quality effects are modified in their expression by the
individual cell types.
The major pigment differences between mesophyll and bundle
sheath chloroplasts are shown in Table I and II. Table II shows
ratios of Chl a to accessory pigments for mesophyll and bundle
sheath preparations (whole leaf data shown in Table I). Bundle
sheath chloroplasts have higher ratios of Chl a to neoxanthin
and Chl b and slightly higher ratios of Chl a to lutein and
carotene. Violaxanthin appears to be equally distributed between
the two chloroplast types. This indicates that bundle sheath
chloroplasts have reduced amounts of the light-harvesting complex but more antennae Chl a associated with the reaction center
complexes.
Light quality does not seem to affect the light-harvesting
pigments in bundle sheath chloroplasts, but pigment ratios indicate that mesophyll chloroplasts in our FR + low R source are
slightly enriched in these complexes. Both mesophyll and bundle
sheath chloroplasts grown in FR + low R light are also enriched
in the pigment violaxanthin.
Data on pigment-protein complexes from Table III can be
correlated with these pigment differences. Bundle sheath chloroplasts are deficient in the light-harvesting complexes, especially
LHPP1 and LHPP3 (Fig. 2), and TLH complexes account for
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LIGHT EFFECTS ON CORN CHLOROPLAST DEVELOPMENT
31
Table III. Pigment-Protein Complexes of Corn Mesophyll and Bundle
Sheath Cells
Per Cent of Total Chl (675 nm)
Complex
Greenhouse
R
FR + low R
Value SD Value SD Value SD
Mesophyll
CPla
CP1
LHPPI
LHPP2
LHPP3
CPa
FP
CPla + CPI
TLH
PSI/IIa
PSI/TLHb
PSII/TLHC
6.3
26.6
18.1
7.8
15.3
9.4
13.6
33.0
41.3
3.5
0.7
0.2
0.4
0.7
0.7
0.5
0.4
0.4
1.5
6.9
27.3
20.7
6.9
13.3
8.9
13.0
34.2
40.9
3.8
0.8
0.2
0.9
0.2
0.3
0.2
0.4
0.1
1
4.4
24.6
26.2
5.3
11.0
13.5
11.8
29.0
42.5
1.1
2.5
0.8
0.2
0.4
0.2
0.4
E
coj
2.1
4581345
0.6
0.3
C) 2
Bundle sheath
CPla
CPI
LHPPl
LHPP2
LHPP3
CPa
FP
CPla + CP1
TLH
PSI/PSII'
5.1
1
6.4 0.6
4.5
1.5
44.1
1.3 38.0 1.4 37.4 2
15.0 0.7
12.3
1.1
16.7
1.3
7.5 0.2
10.5 0.9
3.3 0.4
1
11.1
1
11.9
7.7 0.6
8.4 0.2
8.6 0.5
9.0 0.5
7.1
1.1
12.3
1.9
16.9 3.3
49.2
44.5
41.9
33.7
34.8
27.9
5.8
5.1
4.6
1.4
PSI/TLHb
1.2
1.5
0.2
PSII/TLHC
0.2
0.3
' Ratio of areas of peaks (CPla + CPI)/(CPa) from scans of electrophoresis tube-gels at 675 nm.
b
Ratio of (CPla + CP1)/(LHPPI + LHPP2 + LHPP3).
c Ratio of CPa/(LHPPI + LHPP2 + LHPP3).
approximately 30% of the total Chl. In mesophyll chloroplasts,
light-harvesting complexes account for over 40% of the total Chl.
On the other hand, bundle sheath chloroplasts are enriched in
CP1 complexes, with the total PSI complexes accounting for
approximately 45% of total Chl. In mesophyll chloroplast, PSI
complexes account for slightly more than 30% of total Chl. Of
particular interest is the fact that bundle sheath cells consistently
contain the PSII complex, CPa. Thus, mesophyll chloroplasts
have PSI/PSII ratios of 3 to 3.5, whereas bundle sheath chloroplasts have ratios of 5 to 6. These numbers are ratios of the total
amounts of Chl associated with the respective reaction center
complexes. The ratio of PSI to TLH complexes in bundle sheath
chloroplasts (1.4) is nearly twice that found in mesophyll chloroplasts (0.8), whereas the ratio of PSII to TLH complexes is
equal in both chloroplast types. It should always be borne in
mind that some cross contamination of cell types is possible and
these are only approximate determinations.
The major effects of light quality on pigment-protein complexes is shown in chloroplasts grown under our FR + low R
source. Compared to chloroplasts grown in R or in the greenhouse, FR + low R grown chloroplasts have reduced PSI complexes and enhanced PSII complexes (Fig. 2). TLH complexes
of mesophyll chloroplasts are very slightly enhanced under FR
+ low R light, but FR + low R grown bundle sheath chloroplasts
are deficient in these complexes. Overall, the effect of FR containing a small amount of R is to decrease the ratio of PSI/PSII
and to increase the ratio of PSII/TLH complexes. FR + low R
Far Red
)
Low-Red
46
1
7
Far Red
Low-Red
~~1
~~~
3
N45 6
~~~7
~
7
FIG. 2. Densitometer scan (675 nm) of corn (Golden Bantum OP)
mesophyll and bundle sheath thylakoid pigment-protein complexes separated by tube gel electrophoresis (2). Peaks are identified as follows: (1)
CPIa and (2) CPI (reaction center of PSI), (3) LHPP1, (4) LHPP2, (5)
CPa (reaction center of PSHI), (6) LHPP3 (light-harvesting pigmentprotein monomer and polymeric forms, (7) FP (free pigment) thylakoids
are from GH, R or FR grown plants.
decreases the ratio of PSI/TLH complexes only in the mesophyll.
These results may be further corroborated by membrane polypeptide analysis such as shown in Table IV. Bundle sheath and
mesophyll chloroplasts have nearly equal amounts of the 110
kD (CP1) and 66 kD (CPlapo) proteins, but bundle sheath
chloroplasts have much more of the 21 and 17 kD proteins
associated with PSI. They also have relatively more of a 26 kD
polypeptide (Fig. 3B) which may be associated with light harvesting ofPSI (17). In contrast to this, bundle sheath chloroplasts
have reduced amounts of the 46 kD and 34 kD PSII proteins
but increased amounts of 50 kD and 40 to 43 kD proteins.
Although, 50 kD protein is generally associated with PSII, there
is some contamination by RuBPCO in this area. The lightharvesting proteins (29-31 kD) are also decreased in the bundle
sheaths, but CFl proteins are increased.
Light quality effects are again shown mostly in chloroplast
grown under our FR + low R light source (Fig. 3, A and B; and
Table IV). These effects are reduction of the CPl and CPlapo
proteins and enhancement in the mesophyll of the PSII proteins
(50, 46, and 34 kD). The FR + low R grown bundle sheath
chloroplasts, however, do seem to have slightly more of the 21
and 18 kD proteins associated with PSI, as well as an increased
band at 12 kD (Fig. 3b), perhaps associated with PSI!. Light
harvesting complexes are also much lower in bundle sheath
chloroplasts of FR + low R grown corn. CFl proteins are not
much affected by light quality changes.
Electron micrographs (Fig. 4) show that both greenhouse and
R grown chloroplasts have normal heavy stacking in mesophyll
cells and mostly stroma-type thylakoids in bundle sheath cells.
FR + low R grown chloroplasts have prolamellar bodies in both
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32
ESKINS ET AL.
Table IV. Membrane Polypeptides
Per Cent of Total Protein
R
GH
MESO
BS
MESO
BS
FR+lowR
MESO BS
Plant Physiol. Vol. 77, 1985
mesophyll and bundle sheath cells, with structures similar to the
early stages of greening. Thylakoids in mesophyll cells are mostly
associated as grana, but the grana are very long and consist of
five to six thylakoids only.
DISCUSSION
The mesophyll and bundle sheath cells of Z. mays show an
5.0
2.9
6.6
9.5
6.7
7.4
CP1
and striking structural dimorphism (Fig. 4). The mesoobvious
4.9
1.9
3.8
2.4
3.2
2.5
LHC
contain many highly stacked grana and few
chloroplasts
phyll
2.3
5.4
5.2
2.8
3.4
5.1
CPlapo
bundle sheath chloroplasts are composed
whereas
grains,
starch
8.4
7.3
10.9 11.8 12.4 11.8
CP1 +CPlapo
of stroma lamallae with a few areas of thylakoid appresmainly
1.9
3.2
2.4
2.6
2.1
3.1
(4) (64 kD)
sion and many large starch grains (not evident in our figures).
5.0
4.5
4.7
6.2
5.4
4.3
CFI a(59 kD)
two cell types are similar in structure and function to the
These
9.2
6.6
7.0
7.5
8.1
6.6
CFI , (55 kD)
fraction of the chloroplast. That is, mesophyll cells
subcellular
4.5
3.8
3.7
2.8
4.2
2.8
PSII (50 kD)a
like
grana regions, and bundle sheath cells resemble
more
are
5.1
2.5
2.6
4.5
3.0
4.3
PSII (46 kD)
If one considers only ultrastructure, a comthylakoids.
stroma
7.1
4.9
6.5
5.1
7.2
3.9
40-43 kD
may also be made between mesophyll and normal soyparison
2.9
4.8
3.6
3.7
3.0
3.8
36.5 kD
bean chloroplasts and between bundle sheath and pigment4.1
7.9
3.8
2.9
6.5
5.9
34 kD
mutant chloroplasts. Obviously, there are functional
deficient
4.9
5.2
5.3
5.1
4.3
4.3
32-33 kD
differences resulting from the special requirements
structural
and
9.8
22.5 13.3 18.8 15.5 17.4
LHC (29-31)
that make these comparisons less than
mechanism
the
C4
of
8.4
8.5 11.8
9.3
11.4 11.4
26-28 kD
since bundle sheath cells in other C4
true
is
especially
This
ideal.
8.8
5.2 10.2
7.8
5.1
5.5
21 kD
the prospect of increased
Nevertheless,
agranal.
not
are
species
3.4
7.3
3.1
5.4
5.1
3.4
18 kD
understanding of structure-function relationships by comparison
a Many proteins in this area, especially in bundle sheath, some contam- of such cell types and their response to different light qualities is
intriguing.
ination by RuBPCO.
Our data show that the major differences between mesophyll
and bundle sheath chloroplasts are that bundle sheath chloroplasts, like stroma thylakoids and mutant soybean chloroplasts
(8), have much reduced amounts of those pigments associated
with the light harvesting complexes. The reduction in lightharvesting complexes is primarily due to loss of LHPP1 and
LHPP3. This agrees with previously published data of Anderson
(3). The corresponding loss of membrane polypeptides is in the
29 to 31 kD light-harvesting polypeptides. It is worth noting that
the pigment violaxanthin is evenly distributed between the two
chloroplast types, which may argue for its role being more
involved in membrane formation or regulation, and less associated with specific pigment-proteins of the thylakoids, especially
light-harvesting complexes. In this regard, we have noted in the
past that violaxanthin is always a major constituent of the free
pigment band during gel electrophoresis (19).
A second major difference is that bundle sheath cells, like
stroma thylakoids but unlike mutant pigment-deficient soybeans,
are much enriched in PSI complexes (mainly CPl) and in the
polypeptides associated with PSI (mainly the 21 and 18 kD
proteins). PSII complexes are reduced (CPa), as are the 46 and
34 kD polypeptides. It is important to note, however, that the
bundle sheath chloroplasts still contain several proteins in the 50
kD region, which may also be associated with PSII. Of course, it
is also possible that the 50 kD protein found in bundle sheath
and stroma fraction are not associated with PSII and may be
specialized proteins of the bundle sheath. We do in fact know
that our bundle sheath samples contain measurable amounts of
RuBPCO which contributes to absorption in the 51 kD area. In
work done in the laboratory of Dr. Larry Schrader by Jim
Ostrem, antibodies specific to RuBPCO reacted with our bundle
sheath preparations but gave no reaction with mesophyll prepa0
10
rations. There is still some conflict in the literature about the
Distance, cm
Distne,
occurrence of PSII activity in corn bundle sheath chloroplasts.
FIG. 3. Separation (SDS-PAGE) of membrane polypeptides at 4'C Early reports were very pessimistic (22), but more recent reports
(13) from (a) greenhouse, (b) R, and (c) FR + low R grown corn have concluded that, in the presence of suitable donors and
mesophyll and bundle sheath thylakoids. A, Mesophyll; B, bundle sheath. acceptors, bundle sheath cells have considerable PSII activity
Peaks are identified as follows: (1) CPI, (2) LHC, (3) CPl(apo), 94) 64 (20). In very preliminary work done for us by Drs. R. Herrmann
and P. Westhoff at the Botanisches Institut, University of DuskD, (5) CFI a, (6) CFI ,B, (7) 50.5 kD, (8) 46.5 kD, (9) 40-43 kD, (10)
36.5 kD, (11)34 kD, (12) 32 kD, (13) LHC, (14) 26-28 kD, (15)21 kD, seldorf, West Germany, bundle sheath preparations gave positive
reactions to both PSII 44 and 51 kD antiserum. Thus, we
and (16) 18 kD.
0
cm
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33
LIGHT EFFECTS ON CORN CHLOROPLAST DEVELOPMENT
Mesophyll
Bundle Sheath
Greenhouse
41
Far Red +
Low-Red Light
Red Light
0.5glm
Left:
Mesophyll
Right: Bundle Sheath
/
FIG. 4. Transmission electron micrographs of greenhouse, FR + low R and R grown corn mesophyll and bundle sheath cells. Samples are from
10-d-old first leaf blade.
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ESKINS
ALPlant Physiol. Vol. 77, 1985
343ESISE
conclude that bundle sheath chloroplasts do have PSI1 activity, system to this unequal light distribution. Much work remains to
but the relative amounts of peptides involved in PSII are different be done to fully sort out the effects on both light-harvesting and
in bundle sheath and mesophyll chloroplasts and in stroma and reaction center complexes of various intensities of light alone
grana thylakoids. Perhaps, this different composition may cor- and in the presence of FR.
respond to the a and fl forms of PSI1 noted by Ghirardi and Acknowledgment-The authors wish to express their appreciation to Linda
Melis (12). It is not yet clear which peptide (46 or 50 kD) is the Thurmes and Linda Olson for their help in the analyses of many thylakoid samples.
reaction center, or if both are different types of reaction centers. The authors are especially grateful to Drs. R. Herrmann and P. Westhoff, Botanof
West Germany, for testing our preparaDusseldorf,
It is also not yet totally clear what specific role is played by the ischer Institute, University
using antibodies to PSII peptides (51 and 44 kD) and to Dr. Larry Schrader
34 and 32 kD proteins, but they appear to be more linked with tions
and Jim Ostrem, University of Wisconsin, for testing our preparations using
ET AL.
the 46 kD polypeptide.
Light quality effects in pigment-proteins and membrane polypeptides were very similar in both the greenhouse and R grown
chloroplasts. This was true for both mesophyll and bundle sheath
chloroplasts. The greatest differences were seen in chloroplasts
grown under our FR + low R source. This source has a small
but significant quantity of R that is necessary for greening to
occur. The composition of our FR + low R source leaves open
the question of what is responsible for the changes we see, low R
or FR. In order to answer such questions, we have conducted
experiments on low R and on low R in the presence of FR and
conclude that the effects on chloroplast development are similar
at very low levels of R, especially the effect on the ratio of PSI
toPSII. We conclude that low R in the presence of continuous
FR has repressive effects on PSI proteins during chloroplast
development. Although the amount of R in the FR source is
small, the Chl a/Chl b ratio is as low or lower than R or
greenhouse grown chloroplasts. This parallels the results found
in soybean genotypes (10). FR/low R grown chloroplasts also
have large amounts of violaxanthin in both the mesophyll and
bundle sheaths. The reason for this is not immediately apparent,
but may be related to the immature stage of development under
these light conditions. Most significantly, chloroplasts grown in
this light are enriched in CPa (primarily mesophyll) and depleted
in CPla+CP1. This is reflected in membrane polypeptides by an
increase in the 50, 46, and 34 kD proteins (again primarily in
mesophyll chloroplast) and by a decrease in CPl +CPlapo in
both mesophyll and bundle sheath. There is little change in the
21 and 18 kD proteins in the mesophyll, but the bundle sheath
grown in FR/low R has significantly more of these PSI-associated
PSI in the
proteins. Overall, the result is an increase of PSII toseen
in soymesophyll chloroplasts, which confirms the resultsof Melis
and
beans (10). These results are also similar to those
in plants
Harvey (16) who found enhancement of PSII activity
however, that
grown in a FR-enriched environment. We suspect,the
their results, like ours, are strongly influenced by amount of
R present in their FR source.
The electron micrographs of FR + low R grown chloroplasts
indicate that they are in a preliminary stage of development,
especially the bundle sheath. The stacking shown byasmesophylls
(Fig. 4) are unusual in that most thylakoids occur grana, but
the grana are reminiscent of primary grana seen in early development (14). These thylakoids, however, do not have the high
Chl a/b ratio characteristic of early stages of development. Examounts of
periments with FR sources containing even smallerformed
(Chl
R indicate that less light harvesting complexes are
and
enhancement
of
PSI
b
5
to
but
the
reduction
of
6),
a/Chlproteins are more evident. Since our FR source containsofa
PSII
nm) primarily abhigher proportion of long wave R (680-710
sorbed by PSI, the possibility remains that the reduction of PSI
proteins and enhancement of PSII proteins is a response of the
antibodies to RuBP carboxylase.
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