Cell,Vol. 58, 991-999,
September
8, 1989, Copyright
@ 1989 by Cell Press
Arabidopsisthaliana Mutant That Develops as a
Light-GrownPlant in the Absence of Light
JoanneChory,*t Charles Peto,:t: Rhonda Feinbaum,*
Lee Pratt,§
and Frederick Ausubel*
*Departmentof Genetics
HarvardMedical School and
Departmentof Molecular Biology
MassachusettsGeneral Hospital
Boston,Massachusetts 02114
t PlantBiology Laboratory
TheSalk Institute for Biological Studies
SanDiego,California 92138
t PathologyDepartment
MassachusettsGeneral Hospital
Boston,Massachusetts 02114
§BotanyDepartment
Universityof Georgia
Athens,Georgia 30602
Summary
The signal
transduction
pathways that lead to chlo-
roplastbiogenesis in plants are largely unknown. We
describehere the identification and initial characterizationof a novel genetic locus which fits the criteria
of a regulatory gene located in a central pathway controlling light-mediated development. In the absence of
light,these Arabidopsis thaliana mutants, designated
det1 (de-etiolated
1), constitutively display many char-
acteristics that are light-dependent
in wild-type plants,
including leaf and chloroplast
develop'lnent,
anthocyaninaccumulation, and accumulation
of mRNAs for
several light-regulated
nuclear and chloroplast genes.
Theswitch between dark and light growth
modes thus
appears to
be a programmed step in a developmental
pathwaythat is defined by det1. We suggest a model
where the primary role of light on gene expression
is
mediatedby the activation of leaf development. Further,the recessive nature of the det1 mutation implies
that
there is negative growth control on leaf develop-
mentin dicotyledonous plants in the absence of light.
Introduction
Extrinsicenvironmental signals as well as intrinsic developmentalpathways initiate and control leaf and chloroplastdevelopment in higher plants (Link, 1988; Mullet,
1988).The extrinsic signal, light, is required for chloroplastbiogenesis in all higher plants. In dicotyledonous'
plants,lightsignals also play an important role in the initia-,
tionof primary leaf development, whereas in monocots,
primaryleafdevelopment can take place in the dark in the
absenceof chloroplast development (Dale, 1988). With
the exception of the primary red light photoreceptor,
phytochrome,the biochemistry and molecular biology of
signaltransduction and developmental pathways leading
to leaf and chloroplast development are completely unknown(Smith, 1983; Lagarias, 1985).
Chloroplast development involves the temporally regulated biosynthesis of components of the photosynthetic
apparatus and the carbon reduction cycle and requires
the coordinated expression of both nuclear and chloroplast genes. Phytochrome plays a role in activating nuclear genes that code for chloroplast constituents (Silverthorne and Tobin, 1987); however, the concerted action of
a poorly characterized blue light receptor is required for
optimal photoregulation (Fluhr and Chua, 1986; Senger
and Schmidt, 1986; Silverthorne and Tobin, 1987). Although the levels of some chloroplast mRNAshave been
shown to be positively regulated by red light, in general,
phytochrome appears to playa less significant role in the
regulation of chloroplast genes (Link, 1988). In contrast to
nuclear genes, chloroplast genes are primarily regulated
at the posttranscriptional level (Mullet and Klein, 1987;
Deng and Gruissem, 1987).
In addition to light, the development of chloroplasts is
also regulated by intrinsic developmental signals that controlle.af differentiation (Mullet, 1988; Dale, 1988). This conclusion is based on experiments that demonstrate that inhibition of chloroplast development with herbicides or in
photooxidative mutants does not greatly affect leaf development whereas inhibition of leaf development leads to a
simultaneous inhibition of chloroplast development and
plastid transcription activity (Thompson and Whatley,
1980). Furthermore, only particular leaf-cell types, namely
the mesophyll cells, house matu're chloroplasts, indicating
that cell-specific signals are also important determinants
of chloroplast biogenesis (Dean and Leech, 1982). Finally,
the developmental stage of the chloroplast itself appears
to regulate the expression of nuclear genes coding for
chloroplast-destined proteins (Link, 1988; Mullet, 1988).
For example, in photooxidative mutants of maize or in a variety of plants where chloroplast development is arrested
with an inhibitor, light-regulated nuclear genes are not expressed (e.g., Mayfield and Taylor, 1987), which leads to
the hypothesis that a signal originating from the chloroplast is necessary for optimal transcription of mRNA for
nuclear genes encoding chloroplast proteins (Mayfield
and Taylor, 1987).
Because the regulation of the greening process in
plants is complex, we sought to identify developmental
mutants that would be informative in dissecting the regulatory circuitry involved in transducing both light and developmental signals to chloroplast and nuclear genes.
Since greening is an essential process in plants, we
searched for nonlethal mutants that exhibited abnormal
dark-growth patterns. Dark-grown or "etiolated" dicotyledonous seedlings never make true leaves, have very long
stems and are white in appearance. We reasoned that a
mutation at or near the beginning of the regulatory circuit
controlling leaf and chloroplast development would be
pleiotropic, affecting not only leaf and chloroplast morphogenesis, but also other morphogenic pathways that are
light regulated. In particular, because the effects of light
on dicotyledonous seedling development include inhibi-
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992
tion of stem elongation in addition to leaf expansion and
chloroplast biogenesis, we screened for mutants that appeared "de-etiolated" in the dark, Le., had undergone both
leaf development and stem inhibition.
We report here the isolation and characterization of an
Arabidopsis thaliana mutant designated det1 (de-etiolated).
In the total absence of light, A. thaliana det1 seedlings develop many characteristics which are strictly light-dependent in wild-type seedlings, including leaf and chloroplast
development, anthocyanin accumulation, and constitutive
dark expression of several nuclear and chloroplast lightregulated genes. As such, det1 represents a novel regulatory locus that defines a component which normally couples the onset of leaf and chloroplast development to the
perception of light signals.
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Results
Isolation
of det Mutants
De-etiolated (det) mutants of A. thaliana were identified in
a population of mutagenized M2 generation seeds that
were sown at a density of approximately 3000 seeds per
150 mm petri dish and allowed to grow in complete darkness (see Experimental Procedures for details). Following
7 days of growth in the dark, the plates were scored for
seedlings with short, fat stems and expanded leaves. The
wild-type phenotype for plants grown in the dark is white,
with a long hypocotyl, and no leaf expansion (Fig~re 1).
Some putative det mutants were particularly easy'to identify because in addition to having short stems and expanded leaves, they were also a light purple color, presumably due to the constitutive expression of genes in the
anthocyanin biosynthetic pathway (see below). Out of
100,000 M2 seeds tested, 35 putative de-etiolated mutants were identified. Of these, 20 greened, survived to
maturity, and yielded seed when transferred to white light.
Eight of the 20 tested positive for the det phenotype in the
M3 generation.
The eight det mutants defined two complementation
groups as determined by classical genetic analysis (see
Experimental Procedures). Two alleles of one of these
complementation groups (det1) were analyzed in detail
(Table 1).The wild-type phenotype of the back-crossed F1
and the 3:1 (wild-type: det) segregation in the F2 are consistent with a monogenic recessive trait conferring the
de-etiolated phenotype. This conclusion was supported
by test crosses in which pollen from F1 heterozygotes
(+/det) was crossed to det1 M3 plants, either (det1-1/det11) or (det1-2/det1-2) (Table 1). Both det1 alleles were recessive and both showed similar aberrant development of
true leaves that were purple in the dark; however, the det11 homozygote had slightly better seed set and was therefore used in most subsequent studies.
The det1 homozygotes also had an abnormal phenotype when grown in the light (Figure 1).They were smaller
and paler than wild-type plants grown under the same
light conditions (compare Figure 1C with 10). In addition,
the roots of det1 homozygotes turned green when exposed to light (Figure 1C), while wild-type roots remained
white under the same conditions. When callus cultures
were derived from either shoots or roots of det1 mutant
plants, the callus cultures remained green in sucrosecontaining media lacking cytokinins. This was unlikewildtype root- or shoot-derived callus cultures. Even after
approximately 6 months in culture, the det1 shoot- and
root-derived calli were green (chlorophyll concentrations
were approximately 50 I!g/gm tissue). These resultswere
unexpected because heterotrophically grown cultured
plant cells normally are not pigmented.
det1 Mutations Affect Plastid and Leaf Development
in Dark-Grown Seedlings
The homozygous det1 mutants developed true leavesin
the dark. This was shown by examination of crosssections of det1 leaves from 2 week old dark-grown seedlings (Figure 2). Cross-sections of leaves from light-grown
wild-type A. thaliana plants showed the typical cell architecture of C3 dicotyledonous plants, Le., the leaves
were largely composed of mesophyll cells which harbor
chloroplasts. .(These cells are the primary sites for photosynthetic functions.) On either side of the mesophyll
cells was a layer of nonphotosynthesizing epidermal cells
(Figure 2A). In dark-grown wild-type seedlings (etiolated
seedlings), there was no'secondary leaf development,
and the cotyledons did not expand (data not shown).In
contrast, det1 homozygous mutant plants grown in the
dark had a leaf morphology similar to either wild-typeor
det1 plants that were grown in the light (compare Figure
2A with 2B). The leaf mesophyll cells from dark-grown
det1 seedlings were similarly sized to mesophyll cells
from light-grown det1 plants (about 40 I!m diameter),
though they appeared to be slightly less organized. Because a similar cell architecture to light-grown plantswas
observed for det1 seedlings that weregrownin complete
darkness, we concluded that det1 plants developtrue
leaves in the dark.
We also examined plastid morphology in dark-grown
det1 and wild-type plants. Figure 3A shows the typical
etioplast structure of dark-grown wild-type A. thaliana
seedlings. (The term "etioplast" refers to plastids thatare
Table 1. Results of Crosses with det1-1 Mutant
Cross
Type
det1-1/det1-1
F1
32
0
32
F2
415
110
305
62
29
33
45
45
0
37
0
37
Total
De-Etiolated
Etiolated
x
DET1/DET1
DET1/det1-1
x
DET1-1/det1-1
det1-1/det1-1
Test cross
x
DET1-1/det1-1
det1-1/det1-1
'-
x
det1-2/flet1-2
det1-1/det1-1
x
det2-1/det2-1
LightandLeaf Development in A. thaliana
993
'-
Figure1. Phenotypesof Dark- and light-Grown deM and Wild.:fype A. thaliana Seedlings
(A)Dark-grown
deM;(B) dark-grown wild-type; (C) light-grown deM; (D) light-grown wild-type. The bar corresponds to 1 cm. Note the lack of leaf and
cotyledon
expansionand the extended hypocotyl in the wild-type etiolated seedlings.{B). The deM seedlings are diminutive in size and are paler
incolor(C)thanwild-type grown in the comparable light conditions (D). The rosettes of deM plants reach approximately the same size whether grown
inthelightor in the dark; however, we never observed floral structur~s on dark-grown deM plants.
formedin cotyledons of plants grown entirely in the dark,
"etiolated"plants.) Like etioplasts in other species, those
fromA.thalianawere small (less than 5 Ilm in diameter),
irregularlyshaped (Kirk and Tilney-Bassett, 1978; Leech,
1976),andcontained a central paracrystalline assembly of
tubules,termedthe prolamellar body (e.g., see Leech,
1976).In contrast, plastids in the dark-grown det1 plants
(Figure38) showed clear signs of chloroplast development,as evidenced by the lack of prolamellar bodies in
the plastids, the somewhat larger size and more regu lar
lens shape of the plastid, and the formation of some
bithylakoid membrane structures.
det1 Mutants Bypassa Light Requirementfor
AnthocyaninAccumulationand Seed Germination
Anthocyanins are water soluble, vacuolar pigments responsible for the conspicuous red/purple coloration of
plant leaves and are thought to be involved in protection
Cell
994
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B
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Figure 2. Light Micrographs of 1 11mCross-Sections from Wild-Type
Leaves from Plants Grown in the Light (A) and daM Leaves Grown in
the Dark (B)
c
The organization of the leaf mesophyll cells from dark-grown daM
plants is the same as for either light-grown daM or light-grown wild-type
plants. Chloroplasts can be seen on the perimeters of the mesophyll
cells. Dark-grown wild-type cotyledons, on the other hand, show no leaf
development at all (data not shown).
of the plant from harmful irradiation (Schmelzer et aI.,
1988). Depending on the plant species, anthocyanin accumulation has been shown to be regulated by phytochrome, a blue light receptor, a UV-absorbing receptor, or
by a coaction of light-sensing receptors (Mancinelli and
Rabino, 1978; Rabino and Mancinelli, 1986; Beggs et aI.,
1987). In wild-type A. thaliana, light regulates the transcription of the gene encoding chalcone synthase, the first
committed enzyme in anthocyanin biosynthesis as well
as anthocyanin accumulation (Feinbaum and Ausubel,
1988). Specifically, it appears that the regulation of anthocyanin biosynthetic genes in A. thaliana is primarily
due to a blue light-absorbing photoreceptor (Feinbaum
and Ausubel, unpublished data).
Anthocyanins do not accumulate in etiolated wild-type
Figure 3. Electron
Grown Wild.:fype
Micrographs
Seedlings
of Representative
(A) and
Dark-Grown
Plastids from Dark.
daM (B)
(C) shows a chloroplast from a light-grown daM Imltant plant for com.
parison. Plastids from wild-type seedlings grown in identical lightcon.
ditions appear similar to the plastid shown in (C) and are notshown.
Lightand Leaf Developl'Rentin A. thaliana
995
Table2. Anthocyanin Assays on det1 and Wild-Type Columbia
Grownunder Various Light Regimes
Anthocyanin Content
As30/gm
Light Regime
Wild-Type
det1
Dark
White
Blue
Red
Far-red
ND
0.20
0.14
0.07
0.05
0.43
0.41
0.39
0.37
0.45
% Germination
Wild-Type
det1
20
100
90
91
14
100
100
95
100
97
Anthocyanin
was measured 10 days after seeds were sown.
NO,no anthocyanins were detected.
A
rbcS
1
shown).
.
We also examined the germination frequency of deM
seedswhen grown under different wavelengths of light
(Table2). Wild-type seeds placed in continuous far-red
lightdo not germinate at a high frequency, most likely becauseof conversion of phytochrome to its ~pectrally inactiveform (lagarias, 1985). In deM seeds, however, germinationfrequencies were greater than 95% regardless of
the light conditions used. This result reinforced the hypothesisthat deM plants induced light-regulated functions
independentlyof the state of activity of the photoreceptor
systems.
Nuclearand Chloroplast Light-Regulated
Genes Are
Constitutively Expressed in the Dark in det1 Plants
Thephenotypes of dark-grown deM plants, including leaf
andchloroplast development and anthocyanin accumulation,suggested that deM plants were developing as if they
perceivedlight. To extend this analysis, we examined the
accumulationof mRNAs known to be positively regulated.
bylight in wild-type and deM seedlings grown in the light
anddark. The mRNAs examined included those correspondingto the nuclear genes for chalcone synthase,
chs,(Feinbaum and Ausubel, 1988), the genes for the
smallsubunit of ribulose bisphosphate carboxylase, rbc8
(Krebberset aI., 1988); and the genes for the light-har-
Notethelargeprolamellar protein body in etioplasts from wild-type (A).
Inthe deM mutant, the developing plastid is larger (similar in size to
chloroplastsfrom light-grown deM seedlings), and the early signs of
thylakoid membraneformationcan
spondsto 111m.
be seen (B).The bar in (C) corre-
3
4
.
B
rbcL
+
psbA
+
4
. ..
..
1
A. thaliana seedlings (Table 2); however, as described
above,we observed that deM seedlings had a purplish
huewhen grown in the dark (Figure 1). We examined the
levelsof anthocyanin accumulated in wild-type and deM
plantsgrown under various light regimes (Table 2). Greater
than50-foldthe amount of anthocyanins accumulated in
dark-growndeM plants compared with dark-grown wildtypeseedlings. The levels were similar for dark- and lightgrowndeMplants. The levels of antpocyanin accumulated
indark-growndeM plants were about 2-fold higher than for
light-grownwiJd-typeseedlings. The same results were obtainedusing either white, red, or blue light (Table 2).
Similarresults were observed for deM-2 plants (data not
2
2
3
~
Figure 4. Accumulation of mRNAs for rbeS (a representative nuclear
gene, A) and for rbeL and psbA (representative chloroplast genes, B)
in Light-and Dark-GrownWild.:rypeand deM Mutant Plants
See Table 3 for quantitation of the amount of RNA accumulated. The
lanes are: (1) light-grown wild-type; (2) dark-grown wild-type; (3) lightgrown deM; (4) dark-grown deMoTwo micrograms of total RNAwas
loaded per lane.
vesting chlorophyll a/b binding proteins, cab (leutwiler et
aI., 1986). In all three cases, we observed a high level of
mRNA accumulation in dark-grown deM plants (e.g., Figure 4; see Table 3 for quantitation). These levels were
20- to 50-fold higher than those in dark-grown wild-type
seedlings (Table 3). For the chs gene, the levels of RNA
accumulated in dark-grown deM seedlings were identical
to the levels accumulated in either light-grown deM or
wild-type seedlings. Unlike chs, however, it appeared that
. full expression
of the cab genes involved additional
. regulatory factors besides the elimination of the deM gene
product.
We also examined the levels of expression of four different chloroplast-encoded genes in light- and dark-grown
plants. The RNAs examined were those corresponding to
the genes for the 66 kd chlorophyll apoproteins of photosystem I (psaA-B), the 32 kd as-binding protein of photosystem II (psbA), the large subunit of ribulose bisphosphate carboxylase (rbcl), and 168 rRNA (Mullet and Klein,
1987). These four RNAs were present at low levels in darkgrown wild-type seedlings (Figure 4; Table 3). In contrast,
in dark-grown deM seedlings, these mRNAs were present
at levels indistinguishable from deM or wild-type plants
grown in continuous light for 3 weeks (Table 3; Figure 4).
Cell
996
Table 3. Summary of Differences Exhibited between Dark-Grown Wild-Type and det1 Plants
Leaves
Hypocotyl
Pigments
Germination
Chloroplasts
Gene Expression:a
chs (nuclear)
cab (nuclear)
rbcS (nuclear)
rbcL (chloroplast)
psbA (chloroplast)
psaA-B(chloroplast)
rRNA(chloroplast)
a Gene expression
as outlined
values are expressed
in Experimental
Wild-Type
det1
unexpanded cotyledons
long
absent
expanded cotyledons, leaves
short
20% of light levels
undifferentiated
anthocyan ins
100% of light levels
some differentiation
undetected
1-2
2-5
1-2
1-2
1-2
5
100
25
80
100
100
100
100
as a percent
of the total RNA accumulated
Etiolated Plant Phytochrome Levels Are Unaffected
in det1 Mutants
,
I
"
I. .'
"i~1II1I
"iSI. ",,',g,
'~!PlI~'
'1Ib~;
r'f
in wild-type
plants grown in the light. Data were quantitated
Procedures.
Because suppression of hypocotyl growth and the expression of the cab genes are known to be regulated at least
in part by phytochrome, we examined phytochrome levels
in the deM mutants and in wild-type plants grown in the
dark and light. First, we examined the activity of phytochrome by a dual wavelength spectrophotometric assay
using seedlings grown in total darkness for 5 days (Pratt
et aI., 1984). Dark-grown deM seedlings contained approximately 40% of the levels of photoreversible phytochrome
as wild-type etiolated plants (Average tJ..tJ..A
= 0.00304 for
deM vs. 0.00751 for wild-type; see Experimental Procedures for details). Given differences in the cell and organ
types between dark-grown wild-type and deM seedlings
(Figure 1), we did not consider this difference in phytochrome activity significant. Second, immunoblot analysis
of sodium dodecylsulfate extracts of lyophilized dark-grown
A. thaliana seedlings was performed with a monoclonal
antibody, Pea-25, that is directed to pea phytochrome.
This antibody recognizes a highly conserved epitope, detecting phytochrome apoprotein from a wide variety of
plants whether grown in the dark or in the light (Cordonnier et aI., 1986). This monoclonal detected an equivalent
amount of phytochrome in extracts of dark-grown deM mutants as compared with wild-type, with a monomer size of
approximately 120 kd in each case (data not shown). Phytochrome apoprotein was not detected in either the wildtype or the deM seedlings grown in continuous light (data
not shown), which is consistent with the known reduced
expression of this protein in other plants when they are
grown in the light. We concluded, therefore, that phytochrome aberrations were not responsible for the phenotypes observed in deM mutants.
det1 Gene Product Is Not Required for
Light/Dark Transitions
We were interested in whether the deM gene product acts
solely to affect the primary switch between etiolated and
green growth modes or whether the gene product is also
necessary for the dark repression of light-activated func-
tions during normal light-dark cycles. We addressedthis
question by growing deM and wild-type plants in continuous light f()r 3 weeks, and then moving them to the dark
for 2 days. At that time, we isolated RNA from the dark.
adapted plants and from a set of control plants that had
been left in the light. In other plant systems, it hasbeen
shown that accumulatlon of RNA for nuclear genesinvolved in photosynthesis decreases in the dark (Guilliano
et aI., 1988). We confirmed that observation for wild-type
A. thaliana plants using the rbcS and cab genes asprobes
(Figure 5). When we examined the levels of accumulation
of rbcS and cab mRNAs in dark-adapted det1 mutants,we
observed the same decrease in mRNA levels seeninwildtype plants (Figure 5). This result suggests that the det1
gene product acts at the developmental stage associated
with the initiation of chloroplast biogenesis, ratherto repress the expression of these genes during light/darktran.
sitions.
Discussion
We have shown that the recessive A. thaliana det1 mutation affects
a wide variety
2
cab..
3
of light-regulated
traits, includ.
2
4
..
rbcS +
I
3
4
It
""
Figure 5. Accumulation
Type and deM Plants
of cab and rbcS RNAs in Dark-Adapted Wild.
For each panel, the lanes are: (1) light-grown wild-type; (2) dark.
adapted wild-type; (3) light-grown deM; (4) dark-adapted deMo Five
micrograms
of RNA was loaded per lane.
Light and Leaf Development
997
in A. thaliana
'-
ingleaf development,
organelle morphology, gene expression,and germination (Table 3). Dark-grown deM
seedlingslook like light-grown wild-type plants in every
wayexceptfor the fact that they are not green, a result that
isnotunexpected since one of the steps of chlorophyll biosynthesisrequires light as a substrate. The simplest
modelthat explains these observations is that the wildtypedeMgene product (DEn) is a negative regulator or that
it affectsthe activity of a negative factor which acts in a
signaltransduction pathway that couples leaf and chloroplastdevelopment to light perception. Other regulatory
locihavebeen described that give white sectors on green
leavesor that are deficient in photosynthetic complex assembly(Thompson et aL, 1983; Taylor et aL, 1988; Barkan
etaI., 1986) but, in contrast to deM, none unambiguously
fitsthe criteria for an early regulatory gene in chloroplast
biogenesis.
Since deM mutants also have an aberrant phenotype
whengrown in the light, DEn must also play some role
in light-grown plants. For example, deM roots turn green
when exposed to light, unlike wild-type roots grown in the
sameconditions, and deM plants appear paler and generallylesshardy than wild-type plants grown in the light. The
aberrantphenotype of deM mutants when grown in the
light is mostlikely due to the constitutive expression of
light-regulatedgenes in cell types where these genes are
normallysilent. This latter conclusion is consistent with
the observation that callus cultures derived from deM
rootsor shoots make chlorophyll even in the absence of
cytokinins,conditions where calli are normally not pigmented.We also have preliminary evidence using the chs
geneas a probe for in situ hybridizations on leaf crosssectionsthat the chs gene is aberrantly expressed at high
levelsin leaf mesophyll cells in deM mutants (Chory, unpublisheddata). Therefore, in addition to light regulation,
DET1appears to be involved in tissue-specific gene expression. Possible explanations for these observations
arethat the light- and tissue-specific signal transduction
pathwaysdiverge from some common point after DEn, or
that DET1 is part of a tissue-specific pathway that convergeswith an independent light regulatory pathway on
downstreamtarget genes. In either case, DEn is not simplyrequired for the repression of photosynthesis genes in
the dark because the accumulation of mRNAs for these
genes is properly down-regulated in deM mutant plants
that are grown in the light and subsequently shifted to
darkness.
The gene expression patterns in deM plants indicate
thatthe DET1gene product is a primary regulatorof both
chloroplast and nuclear light-regulated genes during
greeningin A. thaliana. It has been a longstanding question asto whether light-regulated nuclear and chloroplast
gene expression is mediated by signals perceived independently by the two compartments or whether it is
mediatedby a primary signal perceived in one compartment and transduced to the second compartment (e.g.,
Rodermelet aL, 1988). The existence of a single genetic
locus,deM,indicates that whatever primary signals are involved in triggering greening, the pathways that lead to
nuclearand chloroplast gene expression must diverge at
some point after DEn. It is interesting to note that nuclear
and chloroplast genes are regulated by different mechanisms (transcriptional vs. posttranscriptional, respectively), which suggests the existence of additional regulatory
steps after activation of the target genes.
DEn appears to be the primary regulator of chloroplast
genes and the chs, rbcS, and cab genes in A. thaliana.
While all these genes are light-regulated, they are probably activated by different light-induced biosynthetic pathways. For instance, the chs gene of Arabidopsis is regulated primarily by blue light (Feinbaum and Ausubel,
unpublished data), while the cab genes are regulated by
phytochrome (Karlin-Neumann et aL, 1988; Chory and
Ashbaugh, unpublished data). This suggests at least one
common point (DEn) to the red and blue light regulatory
pathways. Alternatively, light may trigger a primary switch
for a developmental pathway associated with leaf development which incorporates aspects of all the pathways suggested above. At present we favor this last hypothesis, as
outlined below.
In wild-type dicotyledonous plants, expression of several "photogenes" (e.g., rbcS, rbcl, etc.) is seen only after
exposure of the plants to light, leading to the natural suggestion that light is required for expression of these genes
(Silverthorne and Tobin, 1987). leaf and chloroplast development also require exposure to light, again suggesting
a control mechanism (Dale, 1988). In the absence of leaf
development, a small, but measurable, induction of some
photogenes is seen after pulses of red or blue light are applied to etiolated plantsc-(e.g., Karlin-Neumann et aL,
1988); however, the levels of mRNA accumulated are only
about 5% of the levels seen in light-grown plants. These
mRNAs do not accumulate to light-grown levels unless
leaves develop. In monocots, the situation is quite different. leaf expansion and the expression of several photogenes occurs at near light levels, even in dark-grown
plants (Baumgartner et aL, 1989; Nelson et aL, 1984).
Based on our analysis of the deM phenotype and from
considerations of monocot development, we suggest the
following model for photoregulation of development in dicotyledonous plants. In wild-type plants, leaf expansion is
absolutely dependent on perception of a light signal, and
expression of photogenes occurs largely after the plant
has embarked on the developmental program represented by leaf expansion. Thus, the light-dependence of
gene expression is largely a reflection of the dependence
of leaf development on light and only partially a direct dependence of transcription on light. The deM mutation is
important because it uncouples the onset of the developmental program from light perception. This serves not
only to define genetically the coupling between light and
leaf development, but allows us to assess the dependence of photogene transcription on light. In the uncoupled state (deM) we find that initial expression of the photogenes depends only on leaf development and not on
light. We propose that in wild-type plants as well, the most
immediate effect of light signal perception is the onset of
leaf development. This does not exclude an additional
qualitative effect by light on the extent of gene expression
once triggered by a developing leaf morphology.
~
I
Cell
998
In summary, we have defined a new locus, deM, a mutation which reveals direct control of gene expression by the
developmental state of the leaf. The description of this locus provides the basis for determining the molecular
mechanism of the control. The identification of deM mu-
or complete darkness
tants also provides the initial genetic tools needed for
reconstruction of the pathways associated with sensory
perception of light-regulated development in plants.
with Toluidine Blue. Thin sections were stained with uranyl acetate and
Experimental
Plant
Growth
Conditions,
and
Genetic
det mutants were isolated following ethyl methane
esis (EMS) of wild-type seeds from the Columbia
and Ogren,
I
1982). The mutants
JII
..Ji
""I'
JlIII'
'~I
~ I,
~1~'
Methods
sulfonate mutagenecotype (Somerville
were back-crossed
into the wild-type
t'OIIU
"..)1'
I
f~u
r'
in Spurr's resin (Hirsch et aI.,
Sections (1 J.lm)were stained
lead citrate, and examined
in a Philips 410LS transmission
electron mi-
c;:roscope.
agarose gels (Ausubel et aI., 1987), and blotted onto Gene Screen Plus
(New England Nuclear) using the manufacturer's
recommendations.
Each lane on the agarose gel contained
1 J.l9of total RNA. The filters
for 0.5 to 2 hr at 60°C in a solution containing 1 M
were prehybridized
studies. In the physiological experiments described here, the wild-type
was the Columbia line. Genetic nomenclature is based on the Third In-
washed
Arabidopsis
Meeting
(East Lansing,
MI, 1987). If not speci-
that the mutant allele number
is 1 (e.g., deM-1).
denatured salmon sperm DNA. For homologous probes, filters were
several times at 65°C in 0.1x SSC (15 mM NaCI, 1.5 mM so-
dium citrate [pH 7.0» and 1% SDS; for heterologous probes, the filters
were washed several times at 65°C in 2x SSC (0.3 M NaCI, 0.03 M so-
The M2 seeds used in this study were produced by soaking approximately 50,000 wild-type seeds on 0.3% (v/v) EMS for 15 hr, followed
dium citrate [pH 7.0», 1% SDS. The filters were exposed to XAR-5 film
by extensive washing with water over a period of 2-4 hr. These
seeds were sown at a density of about 1 plantlcm2. The resulting
and rehybridized
M1
M2
seeds from these M1 plants were harvested in eight separate batches
which were kept separate from each other to minimize the number of
mutant plants that were derived from the same M1 plant. This procedure was repeated
for a separate
batch of 50,000
wild-type
Plants were grown at 20°C under a mixture of fluorescent
II'""U
in acetone, and embedded
1983; K. Miller, personal communication).
NaCI, 1% SDS, 10% dextran sulfate. Hybridizations
with specific
probes were for 12-16 hr in the same solution containing 100 ~g/ml
fied, it is assumed
JII
dehydrated
Columbia
background
before complementation
analysis was performed, and were back-crossed
twice more prior to physiological
ternational
~~
2% glutaraldehyde
in 0.1 M sodium phosphate buffer (pH 7.0),for 1 hr,
rinsed in buffer, post-fixed in 2% osmium tetroxide in buffer for 1.5hr,
Northern
Hybridizations
Isolation of RNA was by a modification of the phenol-SDS method (Ausubel et aI., 1987). RNA was separated in formaldehyde-containing
Procedures
Material,
(on synthetic medium supplemented with 1%
sucrose) was used as the source of material. Tissues were fixed with
seeds.
and incan-
at -70°C
with an intensifying
screen.
The filters were then stripped
with a rDNA probe for normalization
of the RNA load
in each lane. Autoradiograms
for different
exposure times were
scanned with a densitometer. Relative amounts of mRNAs were determined by peak-area measurements,
and relative mRNA levels quantitated are an average of three separate hybridizations.
The DNA probes for nuclear genes used in this study were: cab: a
1.8 kb EcoRI fragment containing the A. thaliana AB180 gene for the
descent lights at an intensity of 200-300 J.lElm2/s. Methods for the
growth of plants in pots, seed harvesting, and cross pollination have
light-harvesting
chlorophyll
protein associated with photosystem II
(Leutwiler et aI., 1986); chs: a 3.8 kb partial Hindlll fragment containing
been described
the A. thaliana
chalcone
synthase gene (Feinbaum and Ausubel,
1988); rbcS: 1.8 kb EcoRI fragment containing the A. thaliana rbcS 1A
(Somerville
and Ogren,
1982). For all of the experi-
on light-grown
plants described, deM-1 and wild-type were
grown side-by-side under the same light and humidity conditions. For
ments
RNA analysis, plants were harvested after 3 weeks of growth in continuous light, prior to bolting and leaf senescence. For seedlings that
were grown in the dark, seeds were imbibed for several hours in 1 mM
gibberellic
acid (Sigma) prior to plating on a minimal salts medium sup-
with 2% sucrose (Lloyd et aI., 1986). Alternatively, the wildtype seeds were germinated in the light for 36-48 hr and then moved
plemented
to the dark. det seeds germinated at 100% in the dark, independent
of gibberellic acid or light treatment. Since the same results were'observed with the deM mutant whether or not we preincubated the seeds
in the light (data not shown), we normally treated the mutant seeds in
the same fashion as wild-type seeds. The plants were harvested after
7 to 10 days of growth in a dark room. A green safelight filter was used
during all dark manipulations.
The growth of plants using specific
wavelengths of light was as follows: blue: General Electric Gemini 500
projector lamps with a Kopp #CS5-60 filter; red: 100 W incandescent
bulbs (Sylvania) with a narrrow band selection filter (50 nm bandwith)
with peak at 660 nm, #S40-650S (Corion Corporation, Holliston, MA);
far-red: 5, 150 W incandescent
bulbs separated by running water for
gene which codes for the small subunit of ribulose bisphosphate carboxylase (Krebbers et aI., 1988); rDNA: a 2.5 kb EcoRI fragment containing an A. thaliana
rDNA gene (Richards and Ausubel, unpublished
probes used for chloroplasts were psaA.B:
data). The heterologous
genes for the photosystem reaction center I polypeptides (66 kd);psbA:
the gene for one of the photosystem II reaction center polypeptides (32
kd); rbcL: the gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase;
and chloroplast
rDNA: the gene for 16S chlo.
roplast rDNA (Mullet and Klein, 1987). The DNA was labeled using either a Boehringer
a specific
Mannheim
nick translation
or random primer kit to
activity of at least 5 x 108 cpm/J.lg DNA.
Phytochrome
Analysis
Seeds were germinated
moved into absolute
Phytochrome
in the light for 2 days, after which they were
darkness
photoreversibility
at 25°C
for an additional
was measured
5 days.
at 664 and 728 nm for
of 325 mg fresh weight, packed gently into a vertical
light-path cuvette with a cross-sectional area of 0.33 cm2in a custom.
tissue samples
built, dual-wavelength
spectrophotometer,
similar to that described by
cooling and filtered through one layer of FR-Perspex (Plexiglass type
FRF 700; Westlakes Plastics Co., Lenni, PA). The outputs of the vari-
Pratt et al. (1984). For immunodetection
of the phytochrome apoprotein, 7 day old, dark-grown A. thaliana seedlings were frozen in liquid
ous light sources, measured at seedling level were as follows: white:
200 J.lElm2/sec; blue: 53.5 J.lElm2/sec; red: 53.5 J.lE/m2/sec; far-red: 26
nitrogen and transferred to a lyophilization
apparatus without being
permitted to thaw at any time. Subsequent extraction of lyophilized tis-
J.lE/m2/s.
sue into SDS sample
Extraction
der), electrophoresis
in 7.5% to 15% linear gradient SDS polyacryl.
amide gels, electrotransfer to nitrocellulose, and immunostaining with
the monoclonal antibody to pea phytochrome, Pea-25, followed byrab-
and Analysis
of Anthocyanin
Pigments
For anthocyanin
determinations, 0.1 g of frozen plant tissue was
ground in 1.5 ml microfuge tube with a disposable pestle, and total
plant pigments were extracted overnight in 0.3 ml of 1% HCI in methanol. After the addition of 0.2 ml of H2O, chlorophyll
was separated
from the anthocyan ins by extraction with an equal volume of chloroform. The quantity of anthocyanins was determined by spectrophotometric
measurements
of the
aqueous:methanol
phase
(As3<rAss7)
buffer (1 ml SDS sample
buffer per 45 mg pow-
bit antibodies to mouse immunoglobulins
and alkaline phosphatase.
conjugated
goat antibodies to rabbit immunoglobulins
was as described elsewhere (Cordonnier et aI., 1986). Pea-25 has been shown
by immunoblot assay to detect phytochrome from a wide variety of dicotyledonous plants, whether
nier et aI., 1986).
grown in the dark or in the light (Cordon-
and normalized to the total fresh weight of tissue used in each sample
(Beggs et aI., 1987; Rabino and Mancinelli,
Light and Electron Microscopy
Plant tissue from 10 day old seedlings
Acknowledgments
1986).
We thank S. Worland, D. Voytas, E. Richards, J. Kooter, T. Delaney, and
grown either
in continuous
light
M. Lawton for helpful discussions
throughout
the course of this work,
'"
Lightand Leaf DevelopmeRt
999
in A. thaliana
M.Timkofor communicating
results prior to publication, E Richards,
E.Tobin,M. Timko, and J. Mullet for providing rDNA, cab, rbcS, and
chloroplastgene clones, J. Fallon for use of E M. facilities,
Doanefor assembling the manuscript.
and C.
This work was supported
by a
grantfrom Hoechst AG to Massachusetts
General Hospital, a grant to
L.P. from the National Science Foundation,
and grants from the
Departmentof Energy, The Samuel Roberts Noble Foundation,
ThePrince Charitable Trusts to J. C. at The Salk Institute.
and
Thecosts of publication of this article were defrayed in part by the
paymentof page charges. This article must therefore be hereby
marked"advertisement" in accordance
with 18 U.S.C. Section 1734
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