Plant Physiol. (1998) 118: 803-815
New Arabidopsis cue Mutants Suggest a Close
Connection between Plastid- and Phytochrome Regulation of Nuclear Gene
Expression1
Enrique López-Juez,
R. Paul Jarvis,
Atsuko Takeuchi,
Anton M. Page, and
Joanne Chory*
Plant Biology Laboratory (E.L.-J., R.P.J., A.T., J.C.), and Howard
Hughes Medical Institute (J.C.), The Salk Institute, 10010 North Torrey
Pines Road, La Jolla, California 92037; and The Salk Institute, 10010 North Torrey
Pines Road, La Jolla, California 92037School of Biological
Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX,
United Kingdom (E.L.-J., A.M.P.)
 |
ABSTRACT |
We searched for new components that
are involved in the positive regulation of nuclear gene expression by
light by extending a screen for Arabidopsis cue
(chlorophyll a/b-binding
[CAB] protein-underexpressed) mutants (H.-M.
Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell
7: 1599-1610). cue mutants display reduced expression
of the CAB3 gene, which encodes light-harvesting
chlorophyll protein, the main chloroplast antenna. The new mutants can
be divided into (a) phytochrome-deficient mutants (hy1
and phyB), (b) virescent or delayed-greening mutants
(cue3, cue6, and cue8),
and (c) uniformly pale mutants (cue4 and
cue9). For each of the mutants, the reduction in
CAB expression correlates with the visible phenotype,
defective chloroplast development, and reduced abundance of the
light-harvesting chlorophyll protein. Levels of protochlorophyllide
oxidoreductase (POR) were reduced to varying degrees in etiolated
mutant seedlings. In the dark, whereas the virescent mutants displayed
reduced CAB expression and the lowest levels of POR
protein, the other mutants expressed CAB and accumulated
POR at near wild-type levels. All of the mutants, with the exception of
cue6, were compromised in their ability to derepress
CAB expression in response to phytochrome activation.
Based on these results, we propose that the previously postulated
plastid-derived signal is closely involved in the pathway through which
phytochrome regulates the expression of nuclear genes encoding plastid
proteins.
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INTRODUCTION |
The assembly of the photosynthetic machinery in developing leaves
of higher plants requires the expression of a set of nuclear and
plastidic genes, the products of which will ultimately function in
chloroplasts (for review, see Mullet, 1988
). The coordinated expression
of these genes is regulated by a number of factors, including light
(Chory, 1991) and a postulated signal through which the nucleus
responds to the functional status of the plastid (Oelmüller et
al., 1986; Mayfield, 1990). In dark-grown angiosperms, plastids develop
into etioplasts, which accumulate protochlorophyllide and POR
(Reinbothe et al., 1996). When plants are exposed to light, profound changes in gene expression occur as the photosynthetic apparatus is assembled and the etioplast develops
into a chloroplast. A specific group of photoreceptors, including
both phytochromes and cryptochromes (Thompson and White, 1991), play an
important role in this transition, but the downstream signaling
components involved are only starting to be understood (Terzaghi and
Cashmore, 1995).
The developing plastid itself appears to play an important role in the
regulation of nuclear gene expression for chloroplast proteins. For
instance, the amount of transcripts for several nuclear-encoded
chloroplast-localized proteins declines very rapidly following
treatments that damage plastid integrity (Oelmüller et al.,
1986). These observations suggest a mechanism by which the nucleus can
sense the physiological status of the organelle. In support of this
hypothesis, Arabidopsis mutants have been isolated in which nuclear
gene expression is partially uncoupled from the status of the plastid
(Susek et al., 1993).
Various approaches have been used to understand mechanistically the
perception and transduction of light signals by plants (Kendrick and
Kronenberg, 1994). Genetic screens have been particularly useful
(Fankhauser and Chory, 1997). Screens for the deregulation of light
responses have identified mutations in the negative elements affecting
light signal transduction, notably the
DET/COP/FUS class (Chory et al.,
1989b; Deng et al., 1992; Miséra et al., 1994). Conversely,
positive elements in the light-signaling pathways initiated by
phytochromes and cryptochromes have been uncovered in screens for
mutants with elongated hypocotyls in various light conditions. These
screens have identified the phytochrome apoprotein and chromophore
biosynthetic genes (HY1, HY2, PHYA,
PHYB, and PHYD), cryptochrome genes
(HY4; for review, see Fankhauser and Chory, 1997), and
downstream elements involved in the control of elongation or flowering,
including ELF3 (Zagotta et al., 1996), FHY1 and
FHY3 (Whitelam et al., 1993), HY5 (Oyama et al.,
1997), PEF1 (Ahmad and Cashmore, 1996), and RED1
(Wagner et al., 1997). With the exception of phyB and the
chromophore mutants hy1 and hy2, none of the
other mutants appears to be defective in phytochrome-regulated gene
expression or chloroplast development (Chory et al., 1989a; Reed et
al., 1994).
In an attempt to identify positive elements specifically involved in
phytochrome signaling to nuclear light-regulated promoters, we devised
a screen based on selection for the underexpression of a
light-regulated promoter, CAB3 (Li et al., 1995). The
CAB family of nuclear genes encode the apoproteins of the
light-harvesting complex of PSII. Following translation on cytoplasmic
ribosomes, the polypeptides encoded (LHCPs) are imported into
chloroplasts and are subsequently integrated into the
thylakoid membrane, where they form the most abundant
chlorophyll-containing complex (Green and Salter, 1996). The expression
of the CAB genes is a marker for chloroplast development and
is tightly controlled by both light (Karlin-Neumann et al., 1988) and
plastid signals. Other factors also control CAB expression,
including a circadian clock (Millar and Kay, 1996), hormones (Flores
and Tobin, 1986; Bartholomew et al., 1991), and Suc levels
(Dijkwel et al., 1997). The interactions between these factors
are complex.
Our previous screen identified mutants at two loci that we named
cue1 and cue2 (Li et al., 1995). cue1
has been analyzed in detail. It is a reticulate mutant with pale-green
mesophyll cells and dark-green bundle-sheath cells aligning the veins
(Li et al., 1995). Characterization of this mutant suggested that
functional CUE1 is required for phytochrome to derepress
CAB expression in the light. This initial screen failed to
identify mutants in the genes encoding the photoreceptors, suggesting
that an expanded screen might be useful. We report the identification
of a series of eight new cue mutants. Two of these mutants
are allelic with the well-characterized photomorphogenetic mutants
phyB and hy1 (Koornneef et al., 1980; Parks and
Quail, 1991; Reed et al., 1993), one is a new allele of
cue1, and the other five identify novel loci. All of these
mutants appear to have defects in chloroplast development. Analysis of
their phenotypes and their responses to red-light pulses suggests a
direct role for a previously postulated plastid-derived factor in the
pathways through which phytochrome controls nuclear gene expression.
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MATERIALS AND METHODS |
Genetic Screen and Methods
We used a previously described Arabidopsis line
expressing both ADH (alcohol
dehydrogenase) and UidA
(
-glucuronidase) genes under the control of a CAB3
promoter (Li et al., 1995). The screening procedure was modified as
follows: Seeds from approximately 6000 ethyl
methanesulfonate-mutagenized plants (M1) were
collected in pools of 200 to 300. M2 seeds
were grown in liquid medium (Murashige-Skoog salt mixture, Gamborg's
vitamin mixture, and 2% Suc) in six-well microtiter plates in batches
of 300 seedlings per well with gentle shaking under 150 µmol photons
m
2 s1 white light for
5 d. The Murashige-Skoog medium was then exchanged for medium
containing 3.5 mM allyl alcohol for 1 h. These
conditions allowed 100% rescue of non-ADH-expressing plants
(R002 mutant, Jacobs et al., 1988); therefore, we predicted that
mutants with only moderate phenotypes would be rescued.
The CUE loci were mapped using previously described
molecular markers (Konieczny and Ausubel, 1993; Bell and Ecker, 1994). DNA polymorphisms were scored in a minimum of 35 F2 mutant seedlings (70 chromatids). Double
mutants were isolated in the F2 progeny of the
respective crosses by searching for novel phenotypes at the
expected frequency (1:16). The assignment was confirmed by the absence
of segregation in the next generation, as well as by the appearance of
similar phenotypes in the progeny of F2 plants identified as single mutants.
Plant Growth and GUS Assays
Plants were grown in plates on synthetic Murashige-Skoog medium
(Murashige-Skoog salt mixture, Gamborg's vitamin mixture, 1% Suc, and
0.8% agar). To ensure uniformity of germination, the plates received a
3-d cold treatment in the dark, followed by 1 h under white light
(approximately 100 µmol photons m2
s1) prior to transfer to darkness or white
light. Except where stated, seedlings were analyzed at 5 d of age.
In experiments in which the whole seedling was harvested, seeds were
sown on sterile filter paper overlaying the medium. GUS activities
(Jefferson, 1987) were measured by fluorescence using
4-methylumbelliferyl -D-glucuronide (GIBCO-BRL) as a
substrate.
Pigments and Protein Analysis
Chlorophylls were extracted in dimethyl formamide at 4°C in the
dark, and the concentration of pigments was calculated according to the
method of Porra et al. (1989). Anthocyanin accumulation was determined
spectrophotometrically as described by Chory et al. (1989b).
Total proteins from greenhouse-grown leaves equivalent to those used
for microscopy or 5-d-old dark-grown seedlings were extracted and
analyzed as described previously (López-Juez and Hughes, 1995) on
PVDF membranes (Merck, Poole, UK); 8 µg of total protein per sample
was used in both cases. Quantitation was performed by blotting a
dilution series of 8.0, 2.5, and 0.8 µg of protein from each sample.
The mouse monoclonal antibodies to LHCPs were a gift from Dr. T. Kunkel
(University of Freiburg, Germany). Antiserum recognizing
Arabidopsis POR was a gift from Drs. K. Apel and G. Armstrong (ETH
Zentrum, Zurich, Switzerland).
Light Treatments and RNA Gel Blots
Plants were grown in 100 µmol photons m2
s1 white light in 16-h photoperiods. For single
light-pulse experiments, seedlings were grown in the dark for 5 d
and treated with the light produced by a tungsten illuminator, filtered
through Nikko DIF-BPF-4 induced transmission filters (Vacuum Optics
Corp., Tokyo, Japan). For red light, the filter had a maximum
A647 and cut-off at 675 nm. The far-red light
had a maximum A745 and cut-on at 713 nm. Total fluences were 1,000 µmol photons m2 for red
(20 µmol photons m2
s1 for 50 s) and 10,000 µmol photons
m2 s1 for far-red light
(130 µmol photons m2
s1 for 75 s). The fluence rate of red
light was measured with a PAR meter and that of far-red was estimated
using the transmission spectra of the filters and assuming a 20%
greater light emission from the tungsten source at 750 than at 650 nm.
For experiments with multiple red pulses, light from fluorescent lamps
(Gro-Lux, Sylvania) was filtered through a colored glass filter (Li et
al., 1995) to provide a narrow-band red peak and attenuated with
neutral filters. Pulses of light consisted of 15 min of light of 1.1 µmol photons m2 s1
each every 6 h for 7 d.
For RNA extraction about 150 mg of seedlings was added to 150 µL of
phenol and 500 µL of extraction buffer (100 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, and 1% SDS) in a
liquid-nitrogen-cooled mortar. The seedlings were ground together with
the frozen buffer and phenol into a paste. After the extract was
transferred to a microcentrifuge tube, it was extracted with 250 µL
of chloroform and spun for 3 min, and the RNA-containing supernatant
was precipitated with an equal volume of 4 M LiCl on ice.
After the sample was centrifuged, the pellet was resuspended in water.
Gel blotting, probe construction, and hybridizations were as previously
described (Chory et al., 1989b). The strength of the radioactive signal was quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Light and Electron Microscopy
Rectangular sections extending from the midvein to the leaf edge
were cut from the middle part of young, greenhouse-grown Arabidopsis
leaves and fixed in 4% formaldehyde plus 3% glutaraldehyde in 0.1 M Pipes buffer, pH 7.2, for 1 h. After the samples
were rinsed in Pipes buffer, they were postfixed for 1 h in 1%
buffered OsO4. The samples were again washed in
buffer, dehydrated in ethanol, and embedded in Spurr's resin following
standard procedures. For light microscopy, 0.5-µm sections were
stained with 1% toluidine blue in 1% sodium tetraborate. Silver
sections were stained with saturated alcoholic uranyl acetate and
Reynold's lead stain and viewed on an electron microscope (model EM
109, Zeiss).
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RESULTS |
Isolation of New cue Mutants
We expanded a previously described mutant screen for identifying
light-insensitive mutants of Arabidopsis that monitors for reduced
expression of a light-regulated promoter, CAB3 (Li et al.,
1995). We screened a mutagenized transgenic line, pOCA108, that is an
adh null mutant (R002) carrying two reporter genes: the
full-length CAB3 promoter fused to the ADH gene
of Arabidopsis and the CAB3 promoter fused to the
Escherichia coli GUS (uidA or GUS)
gene. ADH activity can be selected against using the substrate allyl alcohol (Li et al., 1995). We first determined conditions that would allow the rescue of mutants with only a slight to
moderate deficiency in CAB3 expression. Using these
conditions, we screened ethyl methanesulfonate-mutagenized
M2 seedlings in pools derived from 200 to 300 M1 plants. A total of 125,000 seedlings were screened, with
a minimum of 10 M2 seedlings from each
M1 plant. Mutants containing
trans-acting mutations that reduced CAB3 promoter
activity were distinguished from cis-acting CAB3
promoter mutations using a second reporter, CAB3-GUS.
Of 250 seedlings that survived the allyl alcohol selection, 34 lines
had GUS activity of less than 50% of that of the wild-type pOCA108
parent. The mutants were further divided into six classes based on
their phenotypes. The first class contained three independently isolated mutants with long hypocotyls, and complementation tests showed
them to be alleles of hy1 and phyB. One
reticulate mutant, a new allele of cue1, made up the second
class. The third class (cue4 and cue9) was
uniformly paler than the wild type, with cue9 having a
slightly reticulate phenotype. The fourth class (cue3, cue6, and cue8) was virescent, i.e. young leaves
or recently expanded tissues (including young inflorescence shoots and
the basal margins of older leaves), were pale, whereas more mature
tissues were as green as wild-type tissues. The fifth class of mutants
included lines with a mild reduction in CAB expression
(approximately 50% GUS activity) but no visible phenotype. These will
not be described further here. A final group comprised lines that
segregated albino seedlings in their progeny. These may correspond to
partially dominant albino mutations and were not further investigated.
Figure 1 shows the phenotypes of mature
hy1, phyB, cue3, cue4,
cue6, cue8, and cue9 plants compared
with that of pOCA108. These seven mutants are the focus of the studies
that follow.
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| Figure 1.
Visible phenotypes of the cue
mutants and the pOCA108 wild type. Plants were grown in vitro for
25 d under long-day growth conditions (16 h of light, 8 h of
dark). Smaller plants (cue9, cue6hy1,
cue6cue1-3, cue6cue4, and
cue6cue8) were photographed at higher magnification for
clarity. A, pOCA108 wild type; B, hy1; C,
phyB; D, cue3; E, cue6; F,
cue8; G, cue4; H, cue9; I,
cue1-3; J, cue6hy1; K,
cue6phyB; L, cue6cue1-3; M,
cue6cue4; and N, cue6cue8. Bars in A to
N = 1 cm.
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The seven mutations selected for further study were recessive when
backcrossed to the pOCA108 parent. For each mutant, the pale phenotype
segregated in a manner consistent with a mutation at a single locus
(data not shown). cue3 and cue4 exhibited mild seedling lethality (
2 tests indicated
significant differences from a 3:1 F2 ratio for cue4). Pairwise complementation tests confirmed that these
seven lines define distinct loci (results not shown). We mapped the CUE loci and Table I shows
their chromosomal positions and approximate distance (in centimorgans)
to nearby molecular markers.
We monitored the expression of the endogenous CAB genes, as
well as the genes encoding the ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) in white-light-grown cue
seedlings. Figure 2 shows that all of the
mutations resulted in a moderate reduction of the total amount of
CAB mRNAs. In most cases, although not appreciably for
hy1 and cue4, the RBCS transcripts
were also affected to a comparable extent. The visible phenotype and
reduced gene expression phenotypes were linked. Figure
3 shows the cosegregation of the reduced
CAB expression phenotype with the visible greening defect.
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| Figure 2.
Steady-state mRNA levels for the
CAB and RBCS gene families are reduced in
cue mutant seedlings under white light (WL). Seedlings
were grown for 5 d under 100 µmol photons m2
s1 light in 16-h photoperiods, and harvested for RNA
extraction 5 h into the photoperiod of d 5. RNA gel blots were
hybridized to the corresponding probes and normalized to the signal
from rRNA using a phosphor imager. WT, Wild type.
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| Figure 3.
Cosegregation of the visible and the
CAB underexpression phenotypes of each
cue mutant. After each mutant was backcrossed, the
segregating F2 progeny were scored as mutant or wild type
(WT). Three samples of three to five seedlings each were used for GUS
measurements. For each mutant values are shown relative to the wild
type for the parental line (black bars), wild type-like (striped bars),
and visibly mutant (gray bars) F2 seedlings. For
phyB, seedlings of intermediate length (presumably
heterozygous), not shown here, showed wild-type GUS activities. Error
bars = SD.
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cue Mutants Are Defective in Greening and in the
Expression of Genes for Photosynthetic Proteins
To quantify the CAB expression defect, a time course of
GUS accumulation was performed. A close inspection of Figure
4 shows that the three classes of visible
phenotypes
virescent, yellow-green, and long hypocotylcorrespond to
different patterns of CAB3-driven GUS expression. The
phytochrome mutants showed a general reduction in reporter activity
throughout the period examined, during which time the activity
increased about 2-fold in both the wild type and the mutants. The
slow-greening mutants had a much more pronounced defect early in
development than later, with GUS increasing at least 5-fold over the
period analyzed. cue4, a uniformly pale mutant, showed an
overall defect similar to hy1 or phyB; however, cue9 was intermediate between cue4 and the
slow-greening class.
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| Figure 4.
Kinetics of CAB3 promoter-driven
GUS accumulation in cue mutant seedlings in the light.
Seedlings were grown for 15 d under the conditions described in
Figure 2 and harvested, and GUS activity was measured. The curves are
grouped according to the type of visible phenotype, either wild type
(WT) or long-hypocotyl mutants showing overall reductions in GUS
activity (top), slow-greening mutants showing very low early activities
and subsequent steep increases (middle), and uniformly pale or
reticulate mutants (bottom) appearing similar to those in the top panel
or intermediate between the upper and middle ones. Three samples of 1 to 10 seedlings depending on age are shown; error bars = SD. MU, 4-Methylumbelliferyl
-D-glucuronide; prot, protein.
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We also measured chlorophyll accumulation throughout this same
developmental period. The data presented in Figure
5 show that chlorophyll accumulation
correlates well with the pale phenotypes and the CAB
expression patterns described in Figure 4. Phytochrome-deficient and
uniformly pale mutants showed an increase in chlorophyll content of 2- to 3-fold during this period. In the virescent mutants, chlorophyll
levels increased between 7- and 60-fold. To further analyze this
relationship between CAB expression and chlorophyll accumulation, we examined leaves at two stages of development of the
cue6 mutant (which has the most pronounced virescent
phenotype). Compared with mature cue6 leaves, the youngest,
palest cue6 tissues (leaves less than 5 mm long) had both a
very high chlorophyll a/b ratio, indicative of
relatively low amounts of LHCP (13.1 versus 3.0, the wild-type value
being 3.4), and a greatly reduced level of CAB3-GUS activity
(8% versus 33% of the wild type).
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| Figure 5.
Chlorophyll accumulation is reduced in
cue mutants grown in the light. Total chlorophyll was
measured following extraction from seedlings identical to those used
for GUS accumulation kinetics shown in Figure 4. The seedlings shown
are long-hypocotyl mutants (top), virescent mutants (middle), and
uniformly pale or reticulate mutants (bottom). Each measurement
represents three samples of one to five seedlings; error bars = SD. WT, Wild type.
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To examine a different light-regulated response in the mutants, we
measured anthocyanin accumulation. Anthocyanins accumulated in response
to phytochrome in nonphotosynthetic cells. Figure 6 indicates that anthocyanin was not
reduced in any of the new cue mutants. (Anthocyanin levels
in cue4, although lower in this individual experiment, were
comparable overall to the wild type when analyzed over a series of
ages.) Anthocyanin content was actually higher than wild-type levels in
cue9. This is in contrast to the photoreceptor mutants,
which exhibited a 2- to 4-fold reduction in anthocyanin levels. As
such, the cue mutations appear to affect specifically
chlorophyll accumulation and the expression of photosynthetic genes.
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| Figure 6.
Anthocyanin content of cue mutants
does not correlate with CAB expression or chlorophyll
reduction. Anthocyanin was extracted from seedlings 3.5 d after
the transfer of seeds into light, as described for Figure 2. This time
was shown in preliminary experiments to correspond approximately to the
peak of anthocyanin accumulation in the wild type (WT). Some of the
cue mutants were delayed in their growth, but the
pattern shown here was maintained when examined at d 6. Three samples
are shown; error bars = SD.
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Some cue Mutants Are Affected in Both Basal Levels and
Phytochrome Induction of CAB Expression
Results of many studies have underscored the requirement for
chloroplast integrity (functional chloroplast
transcription/translation) for proper expression of nuclear-encoded
photosynthetic proteins. The reduced photosynthetic gene expression in
the new cue mutants might be due to defects in
photosynthetic physiology or chloroplast development rather than being
a direct consequence of a defect in phytochrome signaling. In an
attempt to discriminate between these possibilities, experiments were
performed in which red-light pulses were given to etiolated seedlings.
The light-pulse treatments can induce (derepress) gene expression, with
only modest changes in seedling development or physiology, more
accurately reflecting the primary effects of photoreceptor activation.
This induction is mediated at least in part by phytochrome
(Karlin-Neumann et al., 1988; Reed et al., 1994).
Figure 7 shows the effect of a single
pulse of red light on the derepression of CAB gene
expression: a 14-fold increase in levels of total CAB mRNAs
in the wild type. As reported previously and as shown in Figure 7,
CAB accumulation was slightly reduced in phyB and
about 80% reduced in hy1 (Chory et al., 1989a; Reed et al.,
1994). In these mutants, the dark basal levels of CAB mRNA
accumulation were similar to the wild type. In contrast, the new
cue mutants differed from the wild type in two ways: First, clear reductions in the basal levels of CAB mRNA in
dark-grown seedlings were observed in the virescent mutants
cue3, cue6, and cue8. Second, moderate
decreases in the ratio of light to basal levels of CAB were
seen in cue3, cue4, cue8, and
cue9 but not in cue6. The amount of
CAB mRNA accumulated in response to the red-light pulse was
reduced in all cases. A far-red pulse was able to reverse the effect of
a red-light pulse about 50% in all genotypes, with the exception of
hy1, indicating involvement of phytochrome.
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| Figure 7.
Induction by a single red-light (R) pulse or
reversion by far-red (FR) light following red light of the accumulation
of CAB mRNA in etiolated cue mutant
seedlings. Five-day-old etiolated seedlings were treated (or not) with
a pulse of light, as indicated in "Materials and Methods," and
returned to darkness for 4 h before harvesting. The number above
each bar represents the derepression by a red-light pulse or the ratio
of red light to the basal dark (D) level. CAB mRNA was
not detected in the cue3 samples. n.d., None detected.
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As a control, the expression of eIF4A, a translation
initiation factor, was shown to be unaffected by the red-light pulse in
the wild type and in the mutants (data not shown). Similar trends were
observed in the cue mutants when GUS accumulation was
monitored by repeated red-light pulses (Fig.
8). Although this assay measures
essentially the same phenomenon as the single pulse treatment, in
preliminary experiments we found it to be a more highly reproducible,
quantitative measurement of the effect of photoreceptor activation.
These studies demonstrated a clear reduction in the basal dark levels
of GUS activity in the virescent mutants and a moderate defect in the
derepression by light of the CAB3 promoter in all of the
mutants except cue6.
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| Figure 8.
Induction by repeated red-light (R) pulses of the
accumulation of CAB3-driven GUS activity in
cue mutant seedlings. A pulse of light of 11 µmol
photons m2 s1 was given for 15 min every
6 h from germination. Otherwise the seedlings remained in the
dark. GUS activity was measured after 7 d, in three samples of six
seedlings each. The number above each genotype represents the
derepression by the pulses or the ratio of GUS activity following a
red-light pulse to the dark (D) value. Error bars = SD. MU, 4-Methylumbelliferyl
-D-glucuronide; prot, protein; WT, wild type;
n.d., none detected.
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cue Mutants Have Defective Plastids
To examine chloroplast development in the cue
mutants, we performed both light and electron microscopic analyses of
leaf sections and assayed amounts of LHCP and POR proteins in
light-grown and etiolated seedlings. Together, the analyses indicate a
correlation among reduced greening, defects in mesophyll structure, and
delayed differentiation of chloroplasts.
Cross-sections of leaves from the phytochrome-deficient and
cue4 mutants (Fig. 9) indicate
a reduced leaf thickness compared with the wild type (Fig. 9, B, C, and
E). In other respects, these leaves appear normal. In contrast,
cue3, cue6, cue8, and cue9 showed areas in which air spaces appear in the palisade mesophyll (Fig.
9, D, F, I, and J). This phenomenon, which is also observed in some
mutants with a reticulate phenotype, has been interpreted to be a
result of reduced growth and division rates of mesophyll cells,
resulting in hollow spaces when the epidermis and vascular bundles
expand (E. Kinsman and K. Pyke, personal communication). The virescent
cue6 mutant showed a gradual transition from abnormal, underdeveloped mesophyll in young leaves to almost wild-type tissue in
the center of mature leaves (Fig. 9, F-H).
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| Figure 9.
Light microscopy of leaf sections of the different
cue mutants. A, Wild type; B, hy1-6.2; C,
phyB-17.6; D, cue3; E,
cue4; F, cue6 young leaf, toward the
margin (mostly pale tissue); G, cue6 young leaf, toward
the midvein (increased greening); H, cue6 mature leaf
(greenest tissue for this mutant); I, cue8; J,
cue9 (section bordering the midvein, to the left). Bar
in A = 100 µm; all panels are to same scale.
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The electron micrographs shown in Figure
10 indicate that the greening defect is
associated with a reduction in both plastid size and the size of the
granal stacks in the various mutants. Although detailed quantitation
was not carried out, Figure 10 shows chloroplasts representative of 10 to 60 recorded chloroplasts (and a much greater number visually
inspected). As previously described (Chory et al., 1989a), thin grana
were found in hy1 (Fig. 10B). The three virescent mutants,
cue3, cue6, and cue8, had the least
developed grana (Fig. 10, D, G, and I). cue4 had the mildest
phenotype and the greatest area of appressed thylakoids (Fig. 10E). The
defect in organelle development was restricted to chloroplasts, since
mitochondria appeared normal in all cases (Fig. 10, insets). The
delayed greening in cue6 appeared to correlate with a
transition from proplastids at the margins of young leaves to mature
chloroplasts in green tissues (Fig. 10, F-H). A similar transition was
observed for cue3 and cue8 leaves (data not
shown). We also noticed that chloroplasts from the cue9
mutant, which exhibits some reticulation in the leaves, were more
differentiated in cells close to the midvein than in the mesophyll
(Fig. 10, compare J and K).
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| Figure 10.
Ultrastructure of plastids from each of the
cue mutants at stages of intermediate greening and at
different stages for cue6. Mitochondria shown for
reference in each case (inset, except for cue6). A, Wild
type; B, hy1-6.2; C, phyB-17.6; D,
cue3, from young leaf, toward midvein; E,
cue4; F, cue6, proplastids from margin of
young leaf; G, cue6, from young leaf toward the midvein
(intermediate greening); H, cue6, chloroplast from
mature leaf; I, cue8, from young leaf, toward midvein;
J, cue9, from mesophyll cell not close to the midvein;
K, cue9, from cell close to central vascular bundle. Bar
in A = 1 µm; all panels are to same scale. Bar in A, inset = 0.5 µm; all insets are to same scale.
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To further assess chloroplast development, the accumulation of LHCP was
quantified by western analysis using total proteins from leaf tissue.
Figure 11A shows that the amounts of
LHCP were reduced in all of the cue mutants to an extent
that was related to the accumulation of chlorophyll (Fig. 5) and the
degree of chloroplast development (Fig. 10).
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| Figure 11.
Accumulation of chloroplast (LHCP) and etioplast
(POR) marker proteins in each cue mutant. Total protein
was extracted initially in SDS-urea solution at 80°C, followed by
acetone precipitation and SDS-PAGE. The top membrane contained 8 µg
per sample of protein from green leaves analyzed with an antibody
against LHCP. The bottom membrane contained the same amount of protein
from etiolated seedlings analyzed with an antiserum against total POR.
WT, Wild type.
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|
To examine whether the reduced dark CAB expression
phenotypes in the virescent cue mutants correlated with a
defect in etioplast development, levels of POR protein were quantified
by western analysis. POR accumulates in etioplasts and is the most
abundant constituent of the prolamellar body. We found a parallel
between POR accumulation and the defect in CAB expression in
etiolated seedlings. For example, Figure 11B shows that there is a
large reduction in POR accumulation in the virescent mutants
cue3, cue6, and cue8, which also show
reduced basal levels of CAB. Virtually no POR was detected
in cue3. Unexpectedly, dark-grown hy1 appeared to
have a slightly lower POR content than phyB, which was
confirmed by a dilution series (results not shown).
Double-Mutant Studies
Genetic epistasis studies were performed to determine whether the
genes defined by the cue mutations (or processes depending on them) play a role in light-signal transduction. We crossed cue6 with representatives of each class of mutants
identified in the screen, including the phytochrome mutants. It should
be noted that the nature of the mutations in the CUE alleles
is unknown; however, the phyB mutation appears to be a null
allele, because no PHYB protein was detected on western blots (E. López-Juez and M. Furuya, unpublished observations). Figure 1
shows the visible phenotypes of the double mutants. cue6hy1
and cue6phyB show the long hypocotyl and petiole defects of
the phytochrome-deficient mutants and the virescent phenotype of
cue6. This argues against a role for CUE6 in
light-controlled cell elongation. However, Table
II shows that, although the
cue6phyB double mutant had delayed greening, the reduction
in chlorophyll levels was not additive with phyB (levels of
29% of wild type would have been expected if these mutations were
additive). In contrast, cue8 appeared to be fully epistatic
to cue6, whereas cue6 and cue1
resulted in fully additive chlorophyll deficiencies. The phenotype of
cue6cue4 appeared by visual inspection to be closer to that
of cue6. This lack of additive effects suggests that the
pathways affected by cue6 and phyB or
cue6 and cue8 (and perhaps cue4) do
overlap at least partially.
View this table:
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|
Table II.
Greening phenotype of double mutants, measured as
microgram chlorophyll per gram fresh weight in 7-d-old seedlings
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DISCUSSION |
The cue Phenotype Defines a Specific
Subset of Chloroplast Development Mutants
We demonstrated in this study that the screen for Arabidopsis
cue mutants can be used to successfully identify mutants in light signaling. However, the cue phenotype probably defines
a relatively broad class of mutants, as indicated by the number of loci
identified and the fact that within related phenotypes we did not find
multiple alleles at a single locus.
We did not anticipate that most of the cue mutants would
have defects in greening, since it has been shown that large reductions in CAB expression do not necessarily result in defective
antenna accumulation (Flackmann and Kuhlbrandt, 1995). Likewise,
reductions in LHCP are not necessarily correlated with defects in
CAB mRNA accumulation (Zhang et al., 1992). That the
cue mutants isolated have visible pale phenotypes suggests
that the loci identified may encode products that function in the
plastid. However, the screen has probably defined a specific subset of
plastid defects, since many pale mutants do not have reduced levels of
CAB. For instance, we obtained several mutants with pale
phenotypes in which CAB expression was very close to normal.
The Arabidopsis pale cress (Reiter et al., 1994),
reticulata (Li et al., 1995), and chlorina3
(Carol et al., 1996) mutants also have very pale or albino phenotypes
but close to wild-type levels of CAB expression. In
addition, Arabidopsis transgenic plants with reduced expression of an
ankyrin-repeat-containing gene possess a phenotype remarkably similar
to that of cue6, and yet CAB expression is normal
in the chlorotic tissues (Zhang et al., 1992). Furthermore, a large
number of mutants that have pale or albino phenotypes have a defect in
carotenoid biosynthesis (Robertson, 1975). None of the cue
mutants so far examined (cue4, cue6, and
cue8) are carotenoid deficient (H.P. Mock and B. Grimm, personal communication). No dark phenotype would be expected of mutants
affected in carotenoid biosynthesis. The cue mutants thus belong to a third class of chloroplast-defective mutants, including, as
extreme cases, Arabidopsis cla1 (Mandel et al., 1996) and
Antirrhinum dag (Chatterjee et al., 1996), which show fully
arrested plastid development and absence of CAB expression.
Fortunately, the fact that the cue mutants described result
only in partial defects in plastid development allows the interaction
between phytochrome and plastid signals to be uncovered. It is also
possible that some of the cue mutations define genes
directly required for the control of CAB expression and that
the greening defect is secondary to this effect.
The cue Phenotype Can Be Explained by
a Close Association between Plastid and Phytochrome Signal
Transduction
A central issue in the characterization of the
cue mutants has been to determine the basis for the
cue phenotype in each case. Three possible interpretations
can be envisaged: (a) CAB expression is affected directly
through a failure to transduce light signals, (b) CAB
expression is affected directly through a regulatory pathway unrelated
to light, or (c) CAB expression is affected indirectly
through a defect in plastid development. Assuming that plastid function
and light-signal transduction are independent, we predicted that
light-signal transduction mutants would have wild-type basal levels of
CAB mRNA, with a defect specifically in derepression of
CAB expression by light pulses.
Conversely, the expectation was that CAB expression would be
derepressed normally by light pulses in plastid function mutants. The
results for two of the five new cue mutants indicated both a
dark phenotype and a light induction defect. This fact, together with
the evidence for defective chloroplast and etioplast development, forced us to revise our basic assumption: plastid function and phytochrome signal transduction may in fact be closely related. Whereas
functional CUE proteins are required prior to the perception of light
pulses, the same proteins or their products must be present during or
after the light pulse for a full response to take place. This leads us
to propose a role for plastids in both the expression of CAB
in the dark and its induction by phytochrome.
Do Plastids Play a Direct Role in Phytochrome Signal
Transduction?
The issue of whether plastid signaling is related to the control
of nuclear gene expression by light has been addressed in the past. It
has been argued that, although light and plastid control of promoter
activity were presumed to be separate processes, part of the overall
effect of light on chloroplast development might be due not to the
direct effect of light on transcription but to the subsequent increase
in plastid-signaling capacity associated with the developing
chloroplast (Mayfield, 1990). One study addressed the possibility of a
direct relation on the control of transcript accumulation using the
albostrians mutant of barley. This nuclear mutation results
in the lack of plastid ribosomes in cells of "white" tissue and in
a very reduced expression of nuclear genes for photosynthetic proteins.
The remaining expression was shown to be light regulated (Hess et al.,
1994), and it was therefore proposed that light regulation was
independent of the plastid signal. However, the fact that the mutant
accumulates some chlorophyll in very pale tissues indicates that the
deficiency in plastid translation is not complete. Since the induction
by light in pale and green tissues was not quantified, the possibility
that a partial defect exists in both plastid function and light
induction cannot be ruled out.
We have previously described evidence suggesting that translationally
functional plastids are required for normal phytochrome derepression of
CAB expression. Growth of seedlings in the dark in the
presence of the organellar protein-biosynthesis inhibitor chloramphenicol results in a gene-expression phenotype very similar to
that observed in cue3, cue4, cue8, and
cue9: the basal level of CAB mRNA is reduced and
the induction by light is also impaired (López-Juez et al.,
1996). In agreement with the features of the previously characterized
plastid signal in mustard (Oelmüller et al., 1986), basal and
light-induced mRNA levels were affected only when organellar
translation was inhibited early but not after 48 h of seedling
growth. When seedlings are grown in the dark on medium containing the
carotenoid-biosynthesis inhibitor norflurazon, CAB
expression is still normally derepressed by a light pulse; however, a
high-fluence, high-intensity pulse capable of driving the synthesis and
subsequent photooxidation of some amount of chlorophyll results in
reduced derepression compared with a low-fluence-rate pulse (E. López-Juez and J. Chory, unpublished results). In the absence of norflurazon it is the high-fluence pulse that leads to
higher CAB expression.
Two alternative models could account for these observations
(López-Juez et al., 1996). In the first, a plastid signal would control the amplitude of the phytochrome-induced signal (or vice versa), with both signals being required simultaneously but acting through separate intermediates. In a second model, the primary target
of the phytochrome-induced signal would be the plastid rather than
nuclear genes such as CAB. This signal, modulating plastid
activity, would subsequently be relayed to the nucleus from the
organelle. Several sites of action for the CUE gene products are possible in both models. If the plastid-derived and
phytochrome-induced signals are distinct but simultaneously required,
we predict that the cue mutations primarily affect plastid
function. This would be the most likely explanation for their altered
etioplast phenotype. If the plastids actually mediate phytochrome
signaling, then the CUE gene products could play a role
either inside the plastid or upstream of it, in a signaling pathway
between phytochrome and the plastid. A direct role for some of the
CUE genes in light signal transduction in spite of their
mutant phenotype in the dark is also a possibility if one assumes a
residual flow through the phytochrome signal transduction system in
dark-grown seedlings. Either model predicts a close interaction between
the phytochrome-induced and plastid-derived signals and could explain
the observed epistasis (lack of additivity) between the phytochrome and
cue mutations.
Similarities and Differences among the cue Mutants
Each mutant described here shows unique features, making it likely
that different primary processes are affected in each case. The
cue3 mutation results in the most dramatic plastid defect, since POR protein levels were barely detectable. CAB
expression was also undetectable in young cue3 seedlings,
although the expression of a control gene (eIF4A) was not
affected. The reduction in CAB expression in cue4
can be ameliorated under lower light (40 µmol photons
m2 s1; data not shown),
and the expression in the dark was also similar to that in the wild
type.
Like cue3, cue6 and cue8 are probably
involved in processes required for normal plastid development, even in
the dark. cue6 is unique because it is the only mutation
that does not reduce the effectiveness of light pulses in derepressing
CAB expression. cue9 shows the highest level of
seedling anthocyanin and the greatest reduction in CAB
derepression by light pulses, making its gene product particularly
interesting. A phenomenon of negative reciprocal control has been
proposed between the separate signal transduction pathways inducing
photosynthetic gene expression and anthocyanin biosynthetic gene
expression (Bowler et al., 1994). The CUE9 gene product
could be an element in the former pathway prior to the source of the
negative crosstalk signal. Only the molecular identification of the
mutated genes will allow the assignment of functions to each of the
CUE genes.
Greening-defective mutants have been widely characterized in other
species, particularly in the cereals. As in Arabidopsis, one abundant
class affects chloroplast structural components or chlorophyll
biosynthesis itself without affecting CAB gene expression (Taylor, 1989; Knoetzel and Simpson, 1991), and another common class is
primarily defective in carotenoid biosynthesis (Robertson, 1975). A
nuclear mutation in the grass Lolium temulentum results in a
slow-to-green phenotype and aberrant plastids (Oughan et al., 1992), but the nature of the mutation and its effect on the expression of genes such as CAB are not known.
A slow-greening phenotype is displayed by cr88, a recently
described Arabidopsis mutant defective in the light regulation of
nitrate reductase (Lin and Cheng, 1997). This mutant does in fact show
reduced CAB expression, as well as a long-hypocotyl phenotype. The mutant maps to a position clearly distinct from any of
the cue mutations described here (C.L. Cheng, personal communication). CR88 appears to play a role upstream of the CUE gene
products, controlling both morphological and photosynthetic gene
expression responses.
The proposed notion that phytochrome regulation of nuclear
photosynthetic gene expression could be a manifestation of the underlying plastid-nuclear-signaling mechanism would explain in a
simple way the multiplicity of regulatory mechanisms observed for the
CAB genes. Remarkably, studies published to date that have
analyzed short promoter elements required for light or plastid response
of photosynthetic genes have so far failed to distinguish between
light- and plastid-responsive sequences (Argüello-Astorga and
Herrera-Estrella, 1996; Bolle et al., 1996). It would be interesting to
assay light-responsive photosynthetic gene expression in cells devoid
of plastids. The existence of plastid-division mutants in which some
cells can be found to be completely devoid of plastids (Robertson et
al., 1995) would make these kinds of experiments possible.
| |
FOOTNOTES |
1
This work was supported by a grant from the U.S.
Department of Energy (no. ER13993 to J.C.). While at The Salk
Institute, E.L.-J. was a fellow of the Spanish Ministry of Education
and of the North Atlantic Treaty Organization. R.P.J. is a long-term fellow of the International Human Frontier Science Program
Organization. J.C. is an associate investigator at the Howard Hughes
Medical Institute.
*
Corresponding author; e-mail chory{at}salk.edu; fax
1-619-558-6379.
Received June 11, 1998;
accepted July 27, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CAB, chlorophyll
a/b-binding protein.
LHCP, light-harvesting chlorophyll protein.
POR, protochlorophyllide
oxidoreductase.
| |
ACKNOWLEDGMENTS |
We thank the members of the Chory laboratory, in particular Drs.
R. Larkin, S. Streatfield, and M. Surpin, as well as Dr. K. Pyke and
Prof. J. Bowyer at Royal Holloway, for their helpful discussions in the
course of this investigation.
| |
LITERATURE CITED |
Ahmad M,
Cashmore AR
(1996)
The pef mutants of Arabidopsis thaliana define lesions early in the phytochrome signaling pathway.
Plant J
10:
1103-1110
[CrossRef][ISI][Medline]
Argüello-Astorga GR,
Herrera-Estrella LR
(1996)
Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways.
Plant Physiol
112:
1151-1166
[Abstract]
Bartholomew DM,
Bartley GE,
Scolnik PA
(1991)
Abscisic acid control of RBCS and Cab transcription in tomato leaves.
Plant Physiol
96:
291-296
[Abstract/Free Full Text]
Bell CJ,
Ecker JR
(1994)
Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.
Genomics
19:
137-144
[CrossRef][ISI][Medline]
Bolle C,
Kusnetsov VV,
Herrmann RG,
Oelmüller R
(1996)
The spinach AtpC and AtpD genes contain elements for light-regulated, plastid-dependent and organ-specific expression in the vicinity of the transcription start sites.
Plant J
9:
21-30
[CrossRef][ISI][Medline]
Bowler C,
Yamagata H,
Neuhaus G,
Chua N-H
(1994)
Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms.
Genes Dev
8:
2188-2202
[Abstract/Free Full Text]
Carol P, Perez P, Begot L, Seyer C, Rocipon M, Mache R (1996)
Transposon tagging of genes important for chloroplast function. 7th
International Conference on Arabidopsis Research (abstract).
Norwich, UK, p 212
Chatterjee M,
Sparvoli S,
Edmunds C,
Garosi P,
Findlay K,
Martin C
(1996)
DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus.
EMBO J
15:
4194-4207
[ISI][Medline]
Chory J
(1991)
Light signals in leaf and chloroplast development: photoreceptors and downstream responses in search of a transduction pathway.
New Biol
3:
538-548
[ISI][Medline]
Chory J,
Peto CA,
Ashbaugh M,
Saganich R,
Pratt LH,
Ausubel FM
(1989a)
Different roles for phytochrome in etiolated and green plants deduced from characterization of Arabidopsis thaliana mutants.
Plant Cell
1:
867-880
[Abstract/Free Full Text]
Chory J,
Peto CA,
Feinbaum R,
Pratt LH,
Ausubel FM
(1989b)
Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light.
Cell
58:
991-999
[CrossRef][ISI][Medline]
Deng XW,
Matsui M,
Wei N,
Wagner D,
Chu AM,
Feldmann KA,
Quail PH
(1992)
COP1, an Arabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a G
homologous domain.
Cell
71:
791-801
[CrossRef][ISI][Medline]
Dijkwel PP,
Huijser C,
Weisbeek PJ,
Chua N-H,
Smeekens SCM
(1997)
Sucrose control of phytochrome A signaling in Arabidopsis.
Plant Cell
9:
583-595
[Abstract]
Fankhauser C,
Chory J
(1997)
Light control of plant development.
Annu Rev Cell Dev Biol
13:
203-229
[CrossRef][ISI][Medline]
Flackmann R,
Kuhlbrandt W
(1995)
Accumulation of plant antenna complexes is regulated by posttranscriptional mechanisms in tobacco.
Plant Cell
7:
149-160
[Abstract]
Flores S,
Tobin EM
(1986)
Cytokinin modulation of Lhcp messenger-RNA levelsthe involvement of post-transcriptional regulation.
Plant Mol Biol
11:
409-415
Green BR,
Salter AH
(1996)
Light regulation of nuclear-encoded thylakoid proteins.
In
B Andersson,
AH Salter,
J Barber,
eds, Molecular Genetics of Photosynthesis.
Oxford University Press, Oxford, UK, pp 75-103
Hess WR,
Muller A,
Nagy F,
Borner T
(1994)
Ribosome-deficient plastids affect transcription of light-induced nuclear genes: genetic evidence for a plastid-derived signal.
Mol Gen Genet
242:
305-312
[CrossRef][ISI][Medline]
Jacobs M,
Dolferus R,
van den Bossche D
(1988)
Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutations of Arabidopsis thaliana (L.) Heynh.
Biochem Genet
26:
105-122
[CrossRef][ISI][Medline]
Jefferson RA
(1987)
Assaying chimeric genes in plants: the GUS gene fusion system.
Plant Mol Biol Rep
5:
387-405
Karlin-Neumann GA,
Sun L,
Tobin EM
(1988)
Expression of light-harvesting chlorophyll a/b-protein genes is phytochrome-regulated in etiolated Arabidopsis thaliana seedlings.
Plant Physiol
88:
1323-1331
[Abstract/Free Full Text]
Kendrick RE, Kronenberg GHM, eds (1994) Photomorphogenesis in
Plants, Ed 2. Kluwer Academic Publishers, Dordrecht, The Netherlands
Knoetzel J,
Simpson D
(1991)
Expression and organisation of antenna proteins in the light-sensitive and temperature-sensitive barley mutant chlorina-104.
Planta
185:
111-123
Konieczny A,
Ausubel FM
(1993)
A procedure for mapping Arabidopsis mutations using codominant ecotype-specific PCR-based markers.
Plant J
4:
403-410
[CrossRef][ISI][Medline]
Koornneef M,
Rolff E,
Spruit CJP
(1980)
Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L.) Heynh.
Z Pflanzenphysiol
100:
147-160
Li H-M,
Culligan K,
Dixon RA,
Chory J
(1995)
CUE1: a mesophyll cell-specific positive regulator of light-controlled gene expression in Arabidopsis.
Plant Cell
7:
1599-1610
[Abstract]
Lin Y,
Cheng CL
(1997)
A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus.
Plant Cell
9:
21-35
[Abstract]
López-Juez E,
Hughes M
(1995)
Effect of blue light and red light on the control of chloroplast acclimation of light-grown pea leaves to increased fluence rates.
Photochem Photobiol
61:
106-111
López-Juez E, Streatfield S, Chory J (1996) Light signals
and autoregulated chloroplast development. In W Briggs, RL
Heath, EM Tobin, eds, Regulation of Plant Growth and Development by
Light, ASPP Symposium Series. American Society of Plant Physiologists,
Rockville, MD, pp 144-152
Mandel MA,
Feldmann KA,
Herrera-Estrella L,
Rocha-Sosa M,
Leon P
(1996)
CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.
Plant J
9:
649-658
[CrossRef][ISI][Medline]
Mayfield SP
(1990)
Chloroplast gene regulation: interaction of the nuclear and chloroplast genomes in the expression of photosynthetic proteins.
Curr Opin Cell Biol
2:
509-513
[CrossRef][Medline]
Millar AJ,
Kay S
(1996)
Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis.
Proc Natl Acad Sci USA
93:
15491-15496
[Abstract/Free Full Text]
Miséra S,
Müller AJ,
Weiland-Heidecker U,
Jürgens G
(1994)
The FUSCA genes of Arabidopsis: negative regulators of light responses.
Mol Gen Genet
244:
242-252
[Medline]
Mullet J
(1988)
Chloroplast development and gene-expression.
Annu Rev Plant Physiol Plant Mol Biol
39:
475-502
[CrossRef][ISI]
Oelmüller R,
Levitan I,
Bergfeld R,
Rajasekhar VK,
Mohr H
(1986)
Expression of nuclear genes as affected by treatments acting on the plastids.
Planta
168:
482-492
[CrossRef][ISI]
Ougan HJ,
Thomas AM,
Thomas BJ,
Roberts PC,
Mutinda C,
Hayward MD,
Dalton SJ
(1992)
Leaf development in Lolium temulentum L.: characterisation of a slow-to-green mutant.
New Phytol
122:
261-272
Oyama T,
Shimura Y,
Okada K
(1997)
The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl.
Genes Dev
11:
2983-2995
[Abstract/Free Full Text]
Parks BM,
Quail PH
(1991)
Phytochrome-deficient hy1 and hy2 long hypocotyl mutants of Arabidopsis are defective in phytochrome chromophore biosynthesis.
Plant Cell
3:
1177
[Abstract/Free Full Text] 1186
Porra RJ,
Thompson WA,
Kriedemann PE
(1989)
Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.
Biochim Biophys Acta
975:
384-394
[CrossRef]
Reed JW,
Nagatani A,
Elich T,
Fagan M,
Chory J
(1994)
Phytochrome A and phytochrome B have overlapping but distinct functions in Arabidopsis development.
Plant Physiol
104:
1139-1149
[Abstract]
Reed JW,
Nagpal P,
Poole DS,
Furuya M,
Chory J
(1993)
Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development.
Plant Cell
5:
147-157
[Abstract]
Reinbothe S,
Reinbothe C,
Lebedev N,
Apel K
(1996)
PORA and PORB, two light-dependent protochlorophyllide-reducing enzymes of angiosperm chlorophyll biosynthesis.
Plant Cell
8:
763-769
[CrossRef][ISI][Medline]
Reiter RS,
Coomber SA,
Bourett TM,
Bartley GE,
Scolnik PA
(1994)
Plant Cell
6:
1253-1264
[Abstract]
Robertson DS
(1975)
Survey of the albino and white endosperm mutants of maize: their phenotypes and gene symbols.
J Hered
66:
67-74
[Free Full Text]
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Abstract
Introduction