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Plant Physiol. (1999) 119: 1033-1040
A Photoperiod-Insensitive Barley Line Contains a Light-Labile
Phytochrome B1
Mamatha Hanumappa2,
Lee H. Pratt,
Marie-Michele Cordonnier-Pratt, and
Gerald F. Deitzer*
Department of Natural Resource Sciences and Landscape
Architecture, University of Maryland, College Park, Maryland 20742 (M.H., G.F.D.); and Department of Botany, University of Georgia,
Athens, Georgia 30602 (L.H.P., M.-M.C.-P.)
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ABSTRACT |
Barley
(Hordeum vulgare L.) is a long-day plant whose flowering
is enhanced when the photoperiod is supplemented with far-red light,
and this promotion is mediated by phytochrome. A chemically mutagenized
dwarf cultivar of barley was selected for early flowering time (barley
maturity daylength response [BMDR]-1) and was made isogenic with the
cultivar Shabet (BMDR-8) by backcrossing. BMDR-1 was found to contain
higher levels of both phytochrome A and phytochrome B in the dark on
immunoblots with monoclonal antibodies from oat (Avena
sativa L.) that are specific to different members of the phytochrome gene family. Phytochrome A was light labile in both BMDR-1
and BMDR-8, decreasing to very low levels after 4 d of growth in
the light. Phytochrome B was light stable in BMDR-8, being equal in
both light and darkness. However, phytochrome B became light labile in
BMDR-1 and this destabilization of phytochrome B appeared to make
BMDR-1 insensitive to photoperiod. In addition, both the mutant and the
wild type lacked any significant promotion of flowering in response to
a pulse of far-red light given at the end of day, and the end-of-day,
far-red inhibition of tillering is normal in both, suggesting that
phytochrome B is not involved with these responses in barley.
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INTRODUCTION |
Barley (Hordeum vulgare L.) is a long-day plant,
requiring daylengths in excess of some critical minimum to flower
(Vince-Prue, 1975 ). The genetics of its sensitivity to
photoperiod are not completely understood, but Yasuda and Hyashi (1980)
have identified four recessive (ea, early
maturity) and three dominant genes at seven loci that influence
flowering in barley. The homozygous recessive
easp,
eac,
eak, and
ea7 genotypes located on chromosomes 3, 4, 5, and 6, respectively, individually confer extreme earliness of
flowering under short daylengths and relative insensitivity to
photoperiod (Gallagher et al., 1991). Genetic studies have indicated
that the eak recessive homozygote located
on chromosome 5 suppresses other maturity loci that influence the time
to heading in the field (Gallagher et al., 1987). In addition,
five major genes and eight quantitative trait loci controlling
flowering have been mapped by restriction fragment-length polymorphism
analysis in a cross between a spring and a winter cultivar of barley
(Laurie et al., 1995). The gene Ppd-H1, which is
located on the short arm of chromosome 2, controls flowering under long
days (>13 h of light), but has no effect under short days (10 h of
light). A second gene, Ppd-H2, is located on the long arm of
chromosome 5 and controls flowering only under short days. The
nomenclature for these genes follows that used for wheat (Law et al.,
1993) and it is unknown whether they are related to any of the
ea loci. Although it is possible that
eak and Ppd-H2 are allelic, this may not be the case, because the Ppd-2 locus in wheat is a dominant gene, whereas eak is recessive.
We have identified an additional early flowering
genotype (BMDR-1) that is a single gene recessive mutant derived from
an M2 diethyl sulfate-treated dwarf genotype
(Deitzer, 1986). It was made isogenic by backcrossing with the
photoperiodically sensitive wild-type cv Shabet (BMDR-8, which is a
shatter-resistant line of the cv Betzes) and selected over nine
generations for normal stature and extreme earliness of flowering. The
BMDR-1 mutant is thought to be allelic to the
eak genotype (L. Gallagher, personal communication).
The addition of FR during the photoperiod promotes the induction of
flowering in barley, which is mediated by phytochrome (Deitzer et al.,
1979; Deitzer, 1983). Addition of FR throughout a long photoperiod (24 h) completely overcomes the delay in flowering caused by short days (12 h) in the wild type, allowing it to flower as early as BMDR-1 (Principe
et al., 1992). Wild-type plants grown under short photoperiods (12 h)
with supplemental FR flower as early as plants grown in long days (24 h) without FR. However, neither flower as early as BMDR-1, which
flowers at the same time in all photoperiods with or without
supplemental FR.
Phytochrome is a proteinaceous pigment that acts through
photointerconversion between an inactive R-absorbing Pr form and a
physiologically active FR-absorbing Pfr form (Smith and
Whitelam, 1990). Arabidopsis expresses five phytochrome
(PHY) genes, PHYA through PHYE
(Sharrock and Quail, 1989; Clack et al., 1994). It is well established
that the gene product of PHYA (phyA) is necessary for
responses mediated by continuous FR (Nagatani et al., 1993; Parks and
Quail, 1993) and that phyB is necessary for responses mediated by continuous R in etiolated seedlings (Lopez-Juez et al.,
1992; Reed et al., 1993). Seed germination is mediated by phyA under limiting light conditions and by phyB
under natural light conditions (Johnson et al., 1994; Shinomura et al.,
1994). It has been reported that phyA is involved in
daylength perception (Weller et al., 1997) and that phyB is
required for enhanced FR level perception in the shade-avoidance and
EOD promotion of internode elongation in light-grown plants (Smith and
Whitelam, 1990; Johnson et al., 1994; Reed et al., 1994). However,
phyB and at least one other species of phytochrome have also
been implicated in the detection of photoperiod and the EOD promotion
of flowering by FR (Halliday et al., 1994).
Recently, Biyashev et al. (1997) have mapped five phytochrome loci in
barley, which were arbitrarily called phy-1, phy-2, phy-3 on
chromosomes 7, 4, and 5, respectively, and phy-4, which is represented
as duplicate loci on chromosomes 2 and 7. It is unknown which
phytochrome genes these encode or whether mutations at any of these
loci affect flowering. However, phy-3 maps to the same
chromosome as eak (Ppd-H2), which may be
allelic to BMDR-1.
Principe et al. (1992) found BMDR-1 to contain about twice the amount
of total phytochrome and to differ from BMDR-8 with respect to two
other proteins of 26 and 85 kD. However, these two proteins, which are
always absent in the mutant genotype, do not decrease in the wild type
in response to FR. The increased level of spectrophotometrically
detectable total phytochrome was found to be largely accounted for by
an increase in phyA (Principe et al., 1992), but nothing was
known about the regulation of phyB levels. The
hy3 mutant in Arabidopsis (Reed et al., 1993), the ma3R mutant in sorghum
(Childs et al., 1992), the ein mutant in Brassica rapa (Devlin et al., 1992), and the lh mutant in
cucumber (Lopez-Juez et al., 1992) all flower early and lack a
light-stable phyB protein. In Arabidopsis and sorghum it has
been shown that the mutation is located in the PHYB gene
(Reed et al., 1993; Childs et al., 1997). The goal of our study is to
establish the nature of the mutation in BMDR-1 and to determine whether
the failure to respond to photoperiod might also be due to a lack of a
functional phyB.
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MATERIALS AND METHODS |
Plant Material
Seeds of barley (Hordeum vulgare L. cv Shabet
[BMDR-8]) and the BMDR-1 mutant were obtained as 2 of 10 isogenic
lines from a breeding program by Dr. Virgil Smail at the Montana State
University (Bozeman) and were maintained by selfing in the greenhouses
at the University of Maryland (College Park).
Growth Conditions
Seeds were surface-sterilized by soaking in a 20% solution of
commercial bleach (2.5% sodium hypochlorite) for 20 min and washing
thoroughly in sterile deionized water. Seeds used for deetiolation
measurements were sown at a density of 25 seeds on wet tissue paper
(Kimpack) in individual plastic trays before being placed into
light-tight aluminum boxes. Seeds used for the flowering experiments
were sown at a density of 25 seeds in individual 1-quart plastic
freezer boxes filled with coarse vermiculite. They were then
subirrigated in plastic trays that held 12 boxes each with
full-strength Hoagland no. 1 solution. All of these seeds were then
placed in a growth chamber in DD at 20°C ± 0.1°C for at least
4 d to ensure uniform germination before transfer to various light
treatments. After 4 d of DD, seedlings were either allowed to
remain in DD or transferred to a chamber with either continuous R,
continuous FR, or 12-h CW photoperiods with or without supplemental FR.
All conditions were maintained at a constant temperature of 20°C ± 0.1°C and 60% ± 5% RH.
CW was supplied by F48T12/CW/VHO fluorescent lamps (GTE-Sylvania,
Danver, MA) and the supplemental FR was provided by F48T12/232/VHO single phosphor fluorescent lamps (GTE-Sylvania). The photosynthetic photon flux (400-700 nm) was maintained at 150 µmol
m 2 s1 under all 12-h
photoperiods, both with and without supplemental FR, and monitored
using a quantum sensor (model L-190SB, Li-Cor, Lincoln, NE). The R was
obtained from GTE-Sylvania F48T12/236/VHO lamps filtered through two
layers of Roscolux no. 823 red cellophane (Kliegle Bros., New York). FR
was produced from F48T12/232/VHO lamps (GTE-Sylvania) filtered through
a one-eighth-inch-thick F-700 black Plexiglas filter (Westlake
Plastics, Lenni Mills, PA). The levels of both were set at 18 µmol
m2 s1 and the spectral
distribution of these fields was monitored using a spectroradiometer
(model Gamma C3, Gamma Scientific, San Diego, CA).
Coleoptile Length and Leaf Opening
The box containing the individual plastic trays with 25 seeds each
was covered with a clear plastic wrap, placed in a growth chamber, and
covered with black cloth for 4 d. At the end of this germination
period, seedlings were exposed to an additional 3 d of continuous
CW, R, or FR, or allowed to remain in DD. Seedlings were removed 3 d after transfer to light and coleoptile lengths were measured in
millimeters with a ruler from the point of attachment of the scutellum
to the tip. Seedling height was measured in millimeters from the
attachment of the scutellum to the tip of the primary leaf. Leaf
opening was measured as the width of the blade in millimeters at the
broadest point of the leaf.
EOD FR Treatment
At the end of the 4-d DD germination period, the trays containing
the boxes with 25 seedlings each were placed in a growth chamber set to
12 h of CW and 12 h of darkness. At the end of each
photoperiod, plants were given a 10-min pulse of R, FR, R followed
immediately by FR, or FR followed immediately by R. Following treatment
for 18 d, plants were moved to a cold room (4°C) and kept in
darkness until measurement. Plants were gently removed from the
vermiculite and the roots and remaining scutellum discarded. We
determined the average fresh weight of the shoots and selected 10 plants that were within ±10% of the mean fresh weight. These were then dissected under a dissecting microscope to determine the
stage of floral development, the length of the inflorescence, and the
number of tillers formed. We repeated all experiments twice and
analyzed the variance data using the SAS system, version 6.12 (SAS
Institute, Cary, NC).
Protein Extraction and Immunoblot Analysis
Seeds sown for the coleoptile measurements were either left in DD
or exposed to an additional 4 d of continuous CW. On the 4th d,
2 g of coleoptile tips or leaves was harvested, frozen immediately, and stored in liquid nitrogen at  80°C. The frozen samples were then extracted according to the method of Wang et al.
(1992), using boiling SDS buffer. They were homogenized with 4 mL of
hot buffer (125 mM Tris-HCl, 4% [w/v] SDS, 10% [v/v]
2-mercaptoethanol, and 20% [v/v] glycerol, pH 6.8). The homogenate
was transferred to a 30-mL centrifuge tube, heated in a boiling water
bath for 5 min, and centrifuged at 16,000g for 10 min. The
supernatant was recovered, clarified at 16,000g for 10 min,
and stored in aliquots at 80°C. We determined the protein content
by the Bradford reaction, using BSA as a standard.
SDS-PAGE was performed as described by Pratt et al. (1986). Prestained,
high-range Mr standards were obtained from
Bio-Rad. The 7.5% gels were transblotted to nitrocellulose according
to the manufacturer's instructions and stained, again according to the
method of Pratt et al. (1986), except that we used
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as the color
substrate. The whole procedure was repeated twice with each sample and
MAb. The blots were air dried and scanned into the Adobe PhotoShop 3.05 software program using a color scanner (model CJ 10, Canon, Japan).
Pea 25 (P-25) is a MAb directed against etiolated pea phytochrome that
recognizes a highly conserved epitope (Cordonnier et al., 1986) and
therefore detects total phytochrome, but does not distinguish between
multiple species. Oat (Avena sativa L.) 22 (O-22) is a MAb
directed against etiolated oat phytochrome and it specifically
recognizes the 124-kD phytochrome apoprotein of phyA
(Cordonnier et al., 1983). GO-5 and GO-7 are specific for a 125- and a
123-kD phytochrome molecular species, respectively, and were developed
against green oat phytochrome (Pratt et al., 1991). Each lane in the
figures represents a separate extract from replicate samples. All MAbs
and the nonimmune mouse IgG used as a control were applied at a 1.0 µg mL1 dilution. Alkaline phosphatase
conjugated to an anti-mouse secondary antibody (Sigma) was applied at a
0.2 µg mL1 dilution.
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RESULTS |
Deetiolation Responses
Because both phytochromes A and B have been reported to play a
role in the photoperiodic induction of flowering (Reed et al., 1993;
Childs et al., 1997; Weller et al., 1997), we examined a number of
de-etiolation responses in both the mutant and wild-type barley that
are known to be mediated by one or the other of these phytochrome
species in other plants. Hypocotyl elongation in dicots and coleoptile
elongation in monocots is strongly inhibited when etiolated seedlings
are exposed to either continuous R, which is mediated primarily by
phyB, or to continuous FR, which is mediated by
phyA. Therefore, dark-grown plants were allowed to remain in DD or transferred to continuous R, FR, or CW for 3 d (Table
I). Coleoptile lengths were not
significantly different in BMDR-1 and BMDR-8 after 7 d of growth
in the dark, and 3 d of continuous CW inhibited elongation to the
same extent in both. Continuous R and FR were less effective than CW;
however, both inhibited coleoptile elongation to the same extent in
both genotypes. All three light treatments significantly inhibited
elongation in both genotypes, but the degree of inhibition was not
significantly different in the mutant when compared with the wild type.
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Table I.
Responses of etiolated seedlings to 3 d of DD,
CW, continuous R (Rc), or continuous FR (FRc)
Seeds were sown and allowed to germinate in the dark for 4 d in a
growth chamber at 20°C. Seedlings were then exposed to an additional
3 d of continuous CW, R, FR, or allowed to remain in DD.
Coleoptile lengths were measured from the point of attachment of the
scutellum to the tip. Seedling height was measured from the attachment
of the scutellum to the tip of the primary leaf. Leaf opening was
measured as the width of the blade in mm at the broadest point of the
leaf. Significance was established by analysis of variance.
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Mesocotyl elongation is also affected by R and FR, but in a manner
opposite to that of the coleoptile. However, unlike wheat, oats, and
sorghum, the mesocotyl in barley does not elongate, and so we examined
the effect of light on leaf blade elongation. We found no significant
effect of 3 d of CW, R, or FR in either the mutant or the wild
type when compared with those left in the dark (Table I, seedling
height). This was true even when seedlings were exposed to 6 d of
light after germination (data not shown). Although BMDR-1 appeared to
be taller than BMDR-8 when grown under continuous R and FR, neither was
significantly taller than the dark controls or CW-grown plants, which
did not differ significantly between the genotypes. On the other hand,
leaf opening was significantly affected by exposure to continuous CW,
R, and FR (Table I, leaf width). After 7 d of growth in DD, the
leaves of both BMDR-1 and BMDR-8 remained very tightly rolled, whereas
growth for 3 d in CW caused these leaves to unroll completely in
both genotypes. Continuous R was much less effective in both; however,
leaf opening was significantly greater than in the dark controls in
both. There was no significant effect of 3 d of continuous FR on
either genotype.
Flowering Responses
There was no enhancement of flowering by a 10-min EOD pulse of FR
in either BMDR-1 or BMDR-8, whether measured as floral stage or as apex
length (Table II). Plants flowered at the
same time regardless of the treatment given. However, the difference in flowering time between the genotypes is very significant under the
given 12-h photoperiods. BMDR-1 is a very-early flowering genotype and
was already in floral stage 4 by d 18, whereas BMDR-8 was still at
stage 2 (Principe et al., 1992). Although flowering was not affected by
these EOD treatments, the number of tillers formed was significantly
affected (Table II). The reduction in the number of tillers following a
10-min pulse of FR was the same in both the mutant and the wild type
and was completely red-light/FR reversible.
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Table II.
Responses of barley plants grown for 19 d
under 12 h photoperiods to 10 min of EOD R, FR, R followed by FR
(R/FR), or FR followed by R (FR/R) light
Seeds were sown in plastic trays and subirrigated with full-strength
Hoagland solution. All seeds were placed in a growth chamber in DD at
20°C ± 0.1°C for 4 d prior to transfer to a growth
chamber set to 12 h of CW and 12 h of dark. At the end of
each photoperiod plants were given a 10-min pulse of R, FR, R followed
immediately by FR, or FR followed immediately by R. Following treatment
for 18 d, the average fresh weight of the shoots was determined,
and 10 plants that were within ±10% of the mean fresh weight
were selected. These were then dissected under a dissecting microscope
to determine the stage of floral development, the length of the
inflorescence, and the number of tillers formed. Significance was
established by analysis of variance. Different letters indicate a
significant difference at P = 0.05.
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Phytochrome Analysis
The 124-kD species of phytochrome in oat, which is detected by
O-22, is known to be a phyA (Cordonnier et al., 1983, 1986), whereas the 123-kD species, which is detected by GO-7, is now thought
to be a phyB (Childs et al., 1997). The 125-kD species, to
which GO-5 binds specifically (Wang et al., 1992), and which was
thought initially not to be either a phyA, phyB,
or phyC (Pratt et al., 1991), seems likely to be a
phyC (R. Wikle and M.-M. Cordonnier-Pratt, unpublished
data). BMDR-1 has an elevated level of total phytochrome, as detected
by P-25 (Fig. 1), and an elevated level
of phyA, as detected by O-22 on western blots. Figure
2 shows that BMDR-1 also contains about
twice as much phyB, as detected by GO-7, when compared with
BMDR-8, but no band was detected by GO-5 in either the mutant or the
wild type. Positive controls with extracts of oat tissue showed normal
binding to GO-5, indicating that epitope seen by GO-5 is missing from
barley.
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| Figure 1.
Western blots of 4-d-old etiolated shoots of
BMDR-1 (left) and BMDR-8 (right) immunostained with the MAbs Pea-25 and
O-22. Each lane was loaded with 105 µg (P-25) or 121 µg (O-22) of
total protein and each represents a separate extract from replicate
samples. IgG, Nonimmune mouse IgG used as the control. Molecular masses
of the protein standards are indicated. The original blots were
digitized at 400 DPI using a Canon CJ-10 color scanner, labeled with
Adobe PhotoShop 4.0, and printed on a Hewlett-Packard LaserJet 4 MV
printer. phy, Phytochrome.
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| Figure 2.
Western blots of 4-d-old-etiolated shoots of
BMDR-1 (left) and BMDR-8 (right) probed with the MAbs GO-7 and GO-5.
Each lane was loaded with 190 µg of total protein and each represents
a separate extract from replicate samples. IgG, Nonimmune mouse IgG
used as the control. Molecular masses of the protein standards are
indicated. The original blots were digitized as in the Figure 1 legend.
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PhyA, although present at high levels in 4-d-old etiolated
seedlings, became undetectable in plant extracts that were exposed to
4 d of continuous CW in both BMDR-1 and BMDR-8 (Fig.
3). PhyB, which is a light
stable as Pfr in all systems so far examined, was present at equal
levels in both the light and the dark in BMDR-8 (Fig.
4, bottom). However, the level of
phyB in BMDR-1 (Fig. 4, top) was decreased to barely
detectable levels in light-grown plant extracts when compared with
extracts from etiolated shoots.
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| Figure 3.
Western blots of 4-d-old-etiolated shoots or
those exposed to 4 d of continuous CW of BMDR-1 (top) or BMDR-8
(bottom) immunostained with O-22. Each lane was loaded with 150 µg of
total protein extracted from 4-day-old-etiolated (lanes DD) or
deetiolated (lanes CW) shoots, and each lane represents a separate
extract from replicate samples. The original blots were digitized as in
the Figure 1 legend.
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| Figure 4.
Western blots of 4-d-old-etiolated shoots or
those exposed to 4 d of continuous CW of BMDR-1 (top) or BMDR-8
(bottom) immunoanalyzed with GO-7. Each lane was loaded with 150 µg
of total protein extracted from 4-d-old-etiolated (DD) or deetiolated
(CW) shoots, and each lane represents a separate extract from replicate
samples. The original blots were digitized as in the Figure 1 legend.
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DISCUSSION |
The early flowering phenotype of BMDR-1 is similar to that in
mutants of other systems that lack a functional phyB. It was our hypothesis that BMDR-1 flowered early, regardless of photoperiod, because it also lacked a functional phyB. However, we found
that the mutant contained a higher level of phyB than the
wild type in the dark (Fig. 2). Levels of different phytochromes in
etiolated tissue may reflect the levels present in seeds, which is
important for germination (Johnson et al., 1994; Reed et al., 1994;
Shinomura et al., 1994). However, it is the level of phytochrome in
green tissue that regulates further growth and development. Figure 3 shows that phyA in barley is a typical light-labile species
in both BMDR-1 and BMDR-8. PhyB is also expressed normally
in the wild type and is a typical light-stable form of phytochrome
(Fig. 4, bottom). However, phyB is significantly reduced in
BMDR-1 in the light (Fig. 4, top). This may be the consequence of a
destabilization of the Pfr form of the phyB protein,
resulting in the phyB of BMDR-1 behaving like a
phyA in the light. It is unknown whether the apparent
destabilization of the phyB protein is a consequence of a
mutation in the PHYB gene in BMDR-1 or results from a
reduced rate of transcription and/or a decrease in message stability in the light.
Recently, the Ma3 maturity gene in sorghum
has been mapped to the PHYB locus, and a premature stop codon has been
identified in the PHYB sequence from the mutant
ma3R (Childs et al.,
1997). This mutant is also insensitive to photoperiod (Childs et al.,
1992), flowering very early in a manner similar to that of BMDR-1. This
result strongly suggests that phyB is involved in the
detection of photoperiod. Therefore, it seems very likely that the
instability of phyB in BMDR-1 could be the reason for its
lack of photoperiod sensitivity. A similar mapping strategy is
currently underway in barley.
PhyA has also been reported to be involved in daylength perception in
Arabidopsis (Johnson et al., 1994; Reed et al., 1994) and in peas
(Weller et al., 1997). The phyA mutant fhy1 was deficient in
sensing an inductive photoperiod, but the phyB mutant
hy3, which flowered earlier than both fhy1 and
the wild type in all photoperiods tested, still responded to an
inductive photoperiod. Although fhy1 seedlings flower at the
same time as the wild type under both short and long days, the
phyA mutant flowers late under 8-h photoperiods extended by
8 h with dim incandescent light, indicating that phyA
is also involved in sensing the photoperiod and that phyA
and phyB may have complementary functions in controlling flowering (Johnson et al., 1994). Using phyA and
phyB overexpressing transgenic Arabidopsis plants, Bagnall
et al. (1995) reported early flowering in the phyA
overexpressor in all daylengths, approaching day neutrality. They found
that, although phyB mutants lacked the EOD regulation of
hypocotyl elongation, they showed normal R/FR reversible EOD regulation
of flowering. On the other hand, the phyB overexpressor
showed promotion of flowering following EOD-R treatment and inhibition
of flowering after an EOD-FR treatment. This is the reverse of what was
found for the wild type. They concluded that both light-labile
(phyA) and light-stable (phyB) phytochromes
interact, and that phytochrome species other than phyA and
phyB (physC, D, or E) may also be
involved in this regulation.
Although both phyA and phyB are expressed at higher levels in
dark-grown mutant plants when compared with the wild type (Figs. 1 and
2), and both decline in the mutant in the light (Figs. 3 and 4),
residual levels of phyA may remain higher in BMDR-1, leading to enhanced sensitivity to photoperiod. ELISA measurements following growth for 7 d in the light (Principe et al., 1992) had measurable levels of phyA, which were significantly higher in BMDR-1
than in BMDR-8. In addition, the decrease in phyB levels in
the light in BMDR-1 may decrease the levels of a floral inhibitor,
which, together with the increase in phyA, would result in
complete day neutrality. However, Weller et al. (1997) reported that
the phyA-mediated promotion of flowering in peas resulted
from a reduction in the synthesis or transport of a floral inhibitor,
suggesting that it acts alone to regulate flowering. The fact that
BMDR-1 is the only mutation that completely lacks any response to both
photoperiod and FR suggests that both phyA and
phyB are required for the normal regulation of flowering. It
further suggests that they act through distinct, but interrelated
pathways.
A marked acceleration of flowering has been noted in Arabidopsis plants
in response to a low R-to-FR ratio and even the early flowering
hy3 mutant shows a slight acceleration of flowering (Whitelam and Smith, 1991; Robson et al., 1993). Using mutants lacking
either phyA or phyB or the double homozygous
recessive hy3/hy2, Halliday et al. (1994) and
Bagnall et al. (1995) have shown that phyB and at least one
other phytochrome species are involved in this response. This
accelerated flowering in response to an EOD-FR treatment has also been
noted in sorghum (Williams and Morgan, 1979). The product of a gene
other than PHYB appears to be required for the perception of
the EOD-FR promotion of flowering, which is absent in both the mutant
and wild-type barley (Table II). The fact that the EOD-FR inhibition of
tillering (Table II) is normal in both the mutant and the wild type
indicates that the promotion of flowering and the inhibition of
tillering are mediated by distinct phytochrome species operating
through separate pathways.
Growth responses, such as the inhibition of coleoptile elongation,
seedling height, and the promotion of leaf opening, did not show any
significant difference in either BMDR-1 or BMDR-8 under different light
conditions (Table I). Although the seedling height was somewhat greater
in BMDR-1 than in BMDR-8 in continuous R and FR, neither was
significantly different from those in DD and CW. This increase in
height was due to elongation of the leaf blade, and not to the
elongation of the mesocotyl, as in other grasses such as oats. The
mesocotyl in barley did not elongate and there appeared to be no effect
on this leaf lade elongation in either genotype. However, there was a
slight (but insignificant) increase in BMDR-1, which may reflect the
increased phytochrome levels in BMDR-1 relative to BMDR-8. The
phyB null mutants in other plant species displayed an
elongated growth pattern, had reduced chlorophyll content, and lacked a
deetiolation response to high-irradiance R (Childs et al., 1991, 1992;
Devlin et al., 1992; Lopez-Juez et al., 1992; Reed et al., 1993).
BMDR-1 did not lack a deetiolation response to high-irradiance R and it
was not visibly less green. It did have slightly narrower leaves than the wild type, as is the case with the sorghum mutant (Pao and Morgan,
1986). Because the levels of phyB decreased in light-grown BMDR-1, and the response to continuous R was the same or slightly greater than in the wild type, either the enhanced level of
phyA was able to compensate for the loss of phyB, or the
residual level of phyB was sufficient to mediate this
response.
| |
FOOTNOTES |
1
This work was supported in part by the Maryland
Agricultural Experiment Station (project no. MD-L-97) to G.F.D. and by
the U.S. Department of Agriculture National Research Initiative
Competitive Grants Program (no. 93-00939) to L.H.P. and M.-M.C.-P.
2
Current Address: Laboratory for Photoperception
& Signal Transduction, Frontier Research Program, RIKEN,
Wako, Saitama 351-01, Japan.
*
Corresponding author; e-mail gd3{at}umail.umd.edu; fax
1-301-314-9308.
Received August 31, 1998;
accepted December 1, 1998.
| |
ABBREVIATIONS |
Abbreviations:
BMDR, barley maturity daylength response.
CW, cool-white fluorescent light.
DD, continuous darkness.
EOD, end-of-day.
FR, far-red light.
MAb, monoclonal antibody.
R, red light.
| |
ACKNOWLEDGMENTS |
We wish to thank Dr. Harry Swartz for the generous use of his
laboratory facilities and Mr. Cen Yiqun and Ms. Jin Ma for their helpful assistance.
| |
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Abstract
Introduction