| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiol. (1999) 120: 491-500 Identification of a cis-Regulatory Element Involved in Phytochrome Down-Regulated Expression of the Pea Small GTPase Gene pra21
Laboratory of Plant Molecular Biology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
The pra2 gene encodes
a pea (Pisum sativum) small GTPase belonging to the
YPT/rab family, and its expression is down-regulated by light, mediated
by phytochrome. We have isolated and characterized a genomic clone of
this gene and constructed a fusion DNA of its 5
Plants use light not only as an energy source for photosynthesis
but also as an environmental signal. When a plant germinates in soil
and is exposed to light after elongating its stem, the stem elongation
is immediately repressed, and the leaves expand, turn green, and
photosynthesis is initiated. Many of these changes are caused by the
regulation of gene expression mediated by photoreceptors such as
phytochrome. A pea (Pisum sativum) small GTPase gene, pra2, which belongs to the YPT/rab family (Nagano et al.,
1993 There are many genes up-regulated by phytochrome, such as
rbcS (Sasaki et al., 1983 We have been interested in the expression of the pra2 gene.
Here we have examined the cis-regulatory element
contributing to light down-regulation of the pra2 gene by
transient assay using a reporter gene and etiolated pea
stems.
The accession number for the sequence reported in this article is
AB007911.
Characterization of the pra2 Gene
Plant Material, Particle Bombardment, and Light Treatment Pea (cv Alaska; Snow Brand Seed, Sapporo, Japan) seeds were soaked in water and sowed in a plastic pot separately. The pot (14 mm in diameter) had a polyethylene net at its lower end and was filled with rock wool and placed in an irrigated vat. Seedlings were grown in the dark for 5 or 6 d at 25°C. A seedling was set horizontally in the bombardment device (model GIE-III, Tanaka Co. Ltd., Sapporo, Japan), which was described in detail by Takeuchi et al. (1992) . To the
growing zone of the etiolated stem (between 0 and 1 cm from the top of
the hook), 1.5- to 3.0-µm gold particles were bombarded once. The
gold particles were coated with a mixture of two kinds of plasmids, a
plasmid containing one of the pra2:LUC constructs and a
plasmid containing a 35S-GUS construct. Five micrograms of each plasmid
was mixed with 2-mg gold particles and suspended in 200 µL of
ethanol. A 4-µL aliquot of the suspension was used for each
bombardment. We performed all of the steps during bombardment in a
darkroom with a dim-green safety light. After bombardment, the plant
was returned to the irrigated vat and incubated in continuous white
light or darkness for 12 h at 25°C. White light was provided by
white fluorescent tubes at an intensity of 70 µmol
m 2 s1 (measured by a
quantum sensor, model LI-190SA, LI-COR, Lincoln, NE).
Measurements of Enzyme Activities A stem of the bombarded pea seedling (between 0 and 1 cm from the top of the hook) was ground in liquid N2 using a chilled mortar and pestle. The powder was dispensed into a microcentrifuge tube and mixed with 300 µL of the buffer consisting of 100 mM potassium phosphate, pH 7.8, 1 mM DTT, 1% Triton X-100, and 1 mM EDTA and then centrifuged at 15,000g at 4°C for 5 min. The supernatant was frozen at 80°C until the enzyme assay was
conducted. LUC assays were performed as described by Miller et al.
(1992) using the Pica Gene LUC assay kit (Wako, Osaka, Japan). Photon emission derived from LUC activity was counted by AUTO LUMAT LB953 (Berthold, Bad Wildbad, Germany). GUS activity was measured
by the method of Jefferson et al. (1987), using
4-methylumbelliferyl  -D-glucuronide
(Wako) as the substrate. Resulting 4-methylumbelliferone concentrations
were determined by Fluoroskan II (Labsystems, Research Triangle
Park, NC). To the reaction mixture, 10% methanol was added to
enhance GUS activity (Kosugi et al., 1990).
4-Methylumbelliferone solutions dissolved in 0.2 M Na2CO3
were used as the standards. Proteins were measured using the
DC Protein Assay kit (Bio-Rad). Background activities from
plants bombarded with gold particles only were subtracted from each LUC
and GUS value. All LUC values were normalized to the corresponding GUS
values. Samples of at least four bombardments were independently
assayed for each construct. The mean values were normalized to that of
the full-length construct (PL1) kept in the dark.
Construction of pra2-LUC Plasmids The plasmids were constructed by four methods as described below.
Extraction of Protein and Immunoblotting Total protein for immunoblotting was extracted by grinding tissue with a mortar and pestle at room temperature with sand together with 0.3 mL of buffer and three stem pieces (between 0 and 1 cm from the top of the hook). The buffer contained 125 mM Tris-HCl, pH 6.8, 6% SDS, and 20% glycerol. The mixture was heated at 100°C for 3 min and centrifuged. The supernatant protein was separated by SDS-PAGE, blotted onto a nitrocellulose membrane, probed with monoclonal IgG against the pra2 protein (Nagano et al., 1995) and goat
anti-mouse IgG conjugated to peroxidase (Bio-Rad), and developed with
an ECL kit (Amersham).
Preparation of Nuclear Extract Nuclear extracts were prepared by a modification of the method described by Ishiguro and Nakamura (1992). Pea seedlings were grown in
darkness for 6 d. For light-treated samples, seedlings were
exposed to white light for 6 h before nuclear extraction. The
upper region of pea epicotyls (1-cm section from the top of the hook,
20 g) was cut into pieces. Then they were homogenized with 250 mL
of homogenization buffer containing 10 mM Pipes-KOH, pH 7.0, 1 M hexylene glycol, 10 mM
MgCl2, 5 mM -mercaptoethanol, 1 mM PMSF, 8 µM pepstatin A, and 2.4 µM leupeptin, in a homogenizer (Hitachi, Tokyo, Japan).
The homogenates were filtered through Miracloth (Calbiochem). Nuclei
were pelleted from the homogenate by centrifugation at
2,700g for 15 min, resuspended in 50 mL of washing buffer
consisting of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20% glycerol,
and 5 mM -mercaptoethanol, and then
centrifuged at 5,200g for 15 min. Washing and centrifugation
were repeated three times. Nuclei were resuspended in 3 mL of nuclear
lysis buffer consisting of 15 mM Hepes-KOH, pH
7.5, 1.25 M KCl, 5 mM MgCl2, 2.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 8 µM pepstatin A, and 2.4 µM leupeptin, with gentle stirring. Particulate
material was pelleted by centrifugation at 5,200g for 15 min
and then at 100,000g for 1 h. The supernatant was
desalted by dialysis. The sample was then centrifuged at
12,000g for 15 min, and the supernatant was collected and
stored at 80°C until the assay was performed. Proteins were
measured using the DC Protein Assay kit (Bio-Rad).
Gel-Retardation Assay The gel-retardation assay was performed by the method described by Shimizu et al. (1996) with some modification. Synthetic oligonucleotides for gel-retardation assay were as follows:
5 -GTCTGAGGATTTTACAGTAATAAAGAAACGA-3(WT1) and
5-TCGTTTCTTTATTACTGTAAAATCCTCAGAC-3 (WT2). The 5 end of a 31-bp
WT1 DNA probe was labeled by incubation with
[ -32P]ATP and T4 polynucleotide kinase and
then hybridized with WT2. The nuclear protein (6 µg) was mixed in
binding buffer (20 µL), which consisted of 20 mM
Tris-HCl, pH 8.0, 50 mM KCl, 0.5 mM EDTA, 15 mM MgCl2, 10% glycerol, 1 mM DTT, 2 µg of poly(dI-dC)-poly(dI-dC), and competitor
DNA as indicated in the figure legends. The unlabeled probe was used as
a wild-type competitor. For mutant competitor, the following
oligonucleotides with a 3-bp substitution were prepared and hybridized:
5-GTCTGAGGcTTTTcCcGTAATAAAGAAACGA-3 (MT1) and 5-TCGTTTCTTTATTACgGgAAAAgCCTCAGAC-3 (MT2). The mutated
nucleotides are indicated in lowercase. The reaction mixture was
preincubated for 5 min. Then, 10,000 cpm of the labeled DNA probe was
added to the reaction mixture. The binding reaction was continued for 15 min at 37°C. Samples were loaded onto a 5% polyacrylamide gel that contained 10% glycerol, 45 mM Tris-borate, and 1 mM EDTA (0.5× TBE buffer) and subjected to electrophoresis
at 4°C. The gel was dried and exposed overnight to an imaging plate
(Fuji Photo Film Co., Kanagawa, Japan). The image was visualized with a
Bio Imaging Analyzer (model BAS2000, Fuji).
Characterization of the Genomic Clone of the pra2 Gene and Determination of Its Transcriptional Start Site The nucleotide sequence of the genomic clone of the pra2 gene is shown in Figure 1. The gene contained two exons and one intron. The deduced amino acid sequences agreed with those of cDNA (Nagano et al., 1993), except that Gly-206 was replaced by Ala. This
may be caused by the fact that the cDNA and genomic libraries were derived from different cultivars. The predicted protein sequences had
the conserved domains for GTP binding (Nagano et al., 1993). Primer-extension analysis revealed that the transcriptional start site
was located 196 nucleotides upstream of the translational start site of
the pra2 gene (Fig. 1). The putative TATA box was located 24 bp upstream from this transcriptional start site. A characteristic
feature of the upstream sequence was the presence of a 113-bp inverted
repeat element (from 1226 to 1114), which displayed 88% sequence
similarity with the upstream region of one of the pea lipoxygenase
genes (lox1:Ps:2; Forster et al., 1994). The PCR and Southern-blot
analyses showed that similar elements were dispersed throughout the
genome of pea plants (data not shown). These results showed that the
element is a family of interspersed DNA elements. Whereas the function
of most repetitive DNAs has not been elucidated, some of them have been
implicated in various cellular functions, such as the regulation of
gene expression (Bureau and Wessler, 1994). However, this element is not involved in the light down-regulated expression as described below,
although further study is required to elucidate the role of this
element in the regulation of gene expression.
Intact Epicotyls Were Necessary for This Transient Assay pra2 mRNA and protein are mainly present in the growing zone of etiolated pea seedlings and disappear upon exposure to light (Yoshida et al., 1993; Nagano et al., 1995). Because we wanted to
introduce a reporter gene into this zone, we examined whether it would
be possible to use a stem section of the growing zone. We examined
whether the pra2 protein in the stem section was stable for
12 h in darkness, as it is in intact seedlings. Immunoblot analysis using a monoclonal antibody against the pra2
protein indicated that the pra2 protein of intact seedlings
was found in darkness (Fig. 2a, lane 1)
and decreased after 12 h of white-light irradiation (lane 2). The
pra2 protein also decreased when the stem was cut and kept
in darkness for 12 h (lane 3). The results indicated that cutting
the stem caused some changes that affected pra2 protein
expression and that the stem section could not be used instead of
intact stems for our experiments. Therefore, we used intact seedlings
to examine reporter-gene expression in a transient assay. Bombardment
of the growing zone of intact seedlings with gold particles, which
might wound plants, did not affect the expression of the
pra2 protein (lane 4).
5 The 93-bp Region Conferred Light Down-Regulated Expression to a Heterologous Promoter We performed gain-of-function 3-deletion analysis to determine
whether this 93-bp region was able to confer light down-regulation to a
non-light-regulated heterologous promoter. We fused several 3-deleted
fragments of the pra2 upstream region to the minimal CaMV
35S promoter (90 from the transcriptional start site, designated as
35S90) and LUC reporter gene (Fig. 4). We
constructed five plasmids containing the 93-bp region, as shown in
Figure 4, and compared the light responsiveness of the reporter-gene
expression in each construct. In the absence of the pra2 5
region, the activity of the reporter gene did not change upon light
irradiation, and light down-regulation was not observed (Fig. 4,
control). The GF1 construct, from which the sequence between the
pra2's own TATA box and the translational start site (24
to +196) was deleted, could not confer down-regulated expression of the
reporter gene. However, the further deleted construct (101 to +196),
GF2, could confer expression. This suggests that the element, which is
located between 101 and 25, may interact with the as-1 sequence of
the CaMV 35S90 promoter. This interaction may determine the light response of the GF1 construct. The other 3-deleted constructs, GF3,
GF4, and GF5, conferred down-regulation of the reporter gene. These
results indicated that the 93-bp region was sufficient to confer light
down-regulated expression to a heterologous promoter, 35S90. As shown
by the activity in light, the basal level decreased with the length of
the fused fragment (from GF1-GF4), suggesting that the length or
sequence affected expression at the basal level.
The 24-bp Region Is Involved in Phytochrome Down-Regulated Expression pra2 expression is down-regulated by phytochrome (Yoshida et al., 1993). To investigate whether a cis element
involved in the down-regulation exists in the 93-bp region, we examined
the effect of a brief red-light irradiation on reporter-gene expression using the PL4 construct. After particle bombardment of the PL4 construct, the plants were irradiated with 2 min of red light and kept
for 12 h in darkness, and the reporter enzyme activity was then
measured. A brief red light repressed the reporter enzyme activity, and
this repression was reversed by far-red irradiation provided
immediately after red-light irradiation (Fig.
5, PL4). In the plants bombarded with the
PL5 construct lacking the 93-bp region, red-light irradiation did not
repress the reporter-enzyme activity (Fig. 5, PL5). These results
suggested that the 93-bp region was necessary for phytochrome
down-regulation.
LS Revealed That the 12-bp Element Mediated Phytochrome Down-Regulated Expression To identify the cis element necessary for phytochrome down-regulation, we conducted LS analysis. We prepared five constructs with 6-bp mutations in the 31-bp region of the PL4A construct (Fig. 5), as shown in Figure 6. The 6-bp mutations correspond to the PstI recognition sequence. We examined the effect of red light on expression using the resulting plasmids. LS2 and LS3 did not show red-light down-regulation (Fig. 6). In particular, the red-light repression was completely abolished in the LS3 construct, suggesting that the core region was located in the mutated region within LS3. The other three constructs, LS1, LS2, and LS5, retained the red-light repression. These results indicated that the phytochrome down-regulation was mediated by the element within the 12-bp region, the sequence of which was GGATTTTACAGT. This element did not show any sequence similarity with the previously reported elements involved in phytochrome- or light-regulated expression.
A Nuclear Factor Specifically Bound to the 12-bp Element in Vitro To examine the presence of nuclear factors such as DNA-binding proteins bound to the 12-bp element specifically, we conducted gel-retardation assays using nuclear extracts from pea epicotyls. As a probe, we prepared a 31-bp synthetic oligonucleotide containing the sequence from672 to 642 (WT; Fig.
7a). Addition of the nuclear extracts
from both etiolated and light-irradiated (6 h) pea plants to the
binding reaction mixture showed the retarded band of the DNA-protein
complex (Fig. 7b, lanes 2 and 3). The signal of etiolated plants was
stronger than that of light-irradiated plants (lanes 2 and 3),
suggesting that the amount of this nuclear factor was higher in the
dark-grown epicotyls than in the light-irradiated plants. This
dark-light difference was observed in three independent extracts. To
test whether the observed binding was specific to the 12-bp element, we
prepared an oligonucleotide in which the adenines in the 12-bp element
were changed to cytosines (MT; Fig. 7a). The band was diminished by
50-fold WT competitor (lanes 4 and 5) but not by MT competitor (lanes 6 and 7). MT competitors in 200- or 400-fold excess could not compete
with labeled probe (lanes 8 and 9). These data indicated that the
observed band was a complex of probe DNA and nuclear factor
specifically bound to the 12-bp element.
We present evidence that the pra2 upstream fragment of
2325 bp from the initiation codon can direct a reporter gene to be expressed at a higher level in darkness than in light in the growing zone of etiolated pea stem. In developing a transient assay system, we
found that cutting the stem drastically decreased the pra2 protein level and reporter-gene expression. The expression of the
reporter gene by our transient assay was analogous to that of the
pra2 gene itself (Yoshida et al., 1993
* Corresponding author; e-mail sasaki{at}agr.nagoya-u.ac.jp; fax 81-52-789-4296. Received December 1, 1998;
accepted March 4, 1999.
Abbreviations: CaMV, cauliflower mosaic virus. LS, linker-scanning. LUC, luciferase.
We thank Drs. H. Mori, K. Yoshida, K. Nakamura, K. Maeo, and S. Shimizu for their technical advice. We also thank Dr. A.T. Jagendorf for discussion. The pBI221-LUC+ plasmid was a kind gift of Dr. Kazuyuki Hiratsuka.
Bruce WB, Deng X-W, Quail PH (1991) A negatively acting DNA sequence element mediates phytochrome-directed repression of phyA gene transcription. EMBO J 10: 3015-3024 [ISI][Medline] Bureau TE, Wessler SR (1994) Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. Plant Cell 6: 907-916 [Abstract]
Carabelli M,
Morelli G,
Whitelam G,
Ruberti I
(1996)
Twilight-zone and canopy shade induction of the Athb-2 homeobox gene in green plants.
Proc Natl Acad Sci USA
93:
3530-3535
Colot V, Robert LS, Kavanagh TA, Bevan MW, Thompson RD (1987) Location of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco. EMBO J 6: 3559-3564 [ISI][Medline] Forster C, Knox M, Domoney C, Casey R (1994) lox1:Ps:2, a Pisum sativum seed lipoxygenase gene. Plant Physiol 106: 1227-1228 [Medline]
Han I-S,
Jongewaard I,
Fosket DE
(1991)
Limited expression of a diverged
Ishiguro S,
Nakamura K
(1992)
The nuclear factor SP8BF binds to the 5
Jefferson RA,
Kavanaugh TA,
Bevan MW
(1987)
GUS fusions: Kehoe DM, Degenhardt J, Winicov I, Tobin EM (1994) Two 10-bp regions are critical for phytochrome regulation of a Lemna gibba Lhcb gene promoter. Plant Cell 6: 1123-1134 [Abstract]
Kosugi S,
Ohashi Y,
Nakajima K,
Arai Y
(1990)
An improved assay for Leu W-M, Cao X-L, Wilson TJ, Snustad DP, Chua N-H (1995) Phytochrome A and phytochrome B mediate the hypocotyl-specific downregulation of TUB1 by light in Arabidopsis. Plant Cell 7: 2187-2196 [Abstract] Miller AJ, Short SR, Hiratsuka K, Chua N-H, Kay SA (1992) Firefly luciferase as a reporter of regulated gene expression in higher plants. Plant Mol Biol Rep 10: 324-337 Nagano Y, Matsuno R, Sasaki Y (1991) Sequence and transcriptional analysis of the gene cluster trnQ-zfpA-psaI-ORF231-petA in pea chloroplasts. Curr Genet 20: 431-436 [CrossRef][ISI][Medline]
Nagano Y,
Murai N,
Matsuno R,
Sasaki Y
(1993)
Isolation and characterization of cDNAs that encode eleven small GTP-binding proteins from Pisum sativum.
Plant Cell Physiol
34:
447-455
Nagano Y,
Okada Y,
Narita H,
Asaka Y,
Sasaki Y
(1995)
Location of light-repressible, small GTP-binding protein of the YPT/rab family in the growing zone of etiolated pea stems.
Proc Natl Acad Sci USA
92:
6314-6318
Nagi N, Tsai F-Y, Coruzzi G (1997) Light-induced transcriptional repression of the pea AS1 gene: identification of cis-elements and trans-factors. Plant J 12: 1021-1034 [CrossRef][ISI][Medline] Neuhaus G, Bowler C, Hiratsuka K, Yamagata H, Chua N-H (1997) Phytochrome-regulated repression of gene expression requires calcium and cGMP. EMBO J 16: 2254-2264 Okubara PA, Williams SA, Doxsee RA, Tobin EM (1993) Analysis of genes negatively regulated by phytochrome action in Lemna gibba and identification of a promoter region required for phytochrome responsiveness. Plant Physiol 101: 915-924 [Abstract] Puente P, Wei N, Deng X-W (1996) Combinatorial interplay of promoter elements constitutes the minimal determinants for light and developmental control of gene expression in Arabidopsis. EMBO J 15: 3732-3743 [ISI][Medline] Sasaki Y, Sakihama T, Kamikubo T, Shinozaki K (1983) Phytochrome-mediated regulation of two mRNAs, encoded by nuclei and chloroplasts of ribulose-1,5-bisphosphate carboxylase/oxygenase. Eur J Biochem 133: 617-620 [ISI][Medline] Shimizu S, Itoh Y, Yamazaki K (1996) Temperature-dependent increase in the DNA-binding activity of a heat shock factor in an extract of tobacco cultured cells. Plant Mol Biol 31: 13-22 [CrossRef][ISI][Medline] Takeuchi Y, Dotson M, Keen NT (1992) Plant transformation: a simple bombardment device based on flowing helium. Plant Mol Biol 18: 835-839 [CrossRef][Medline] Terzaghi WB, Cashmore AR (1995) Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol 46: 445-474 [CrossRef][ISI]
Thomas MS,
Flavell RB
(1990)
Identification of an enhancer element for the endosperm-specific expression of high molecular weight glutenin.
Plant Cell
2:
1171-1180
Tonoike H, Han I-S, Jongewaard I, Doyle M, Guiltinan M, Fosket DE (1994) Hypocotyl expression and light downregulation of the soybean tubulin gene, tubB1. Plant J 5: 343-351 [Medline]
Weatherwax SC,
Williams SA,
Tingay S,
Tobin EM
(1998)
The phytochrome response of the Lemna gibba NPR1 gene is mediated primarily through changes in abscisic acid levels.
Plant Physiol
116:
1299-1305
Williams SA, Weatherwax SC, Bray EA, Tobin EM (1994) NPR genes, which are negatively regulated by phytochrome action in Lemna gibba L. G-3, can also be positively regulated by abscisic acid. Plant Physiol 105: 949-954 [Abstract]
Yoshida K,
Nagano Y,
Murai N,
Sasaki Y
(1993)
Phytochrome-regulated expression of the genes encoding the small GTP-binding proteins in peas.
Proc Natl Acad Sci USA
90:
6636-6640
Copyright Clearance Center: 0032-0889/99/120//10
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
 |
|
  M. E. Hudson and P. H. Quail Identification of Promoter Motifs Involved in the Network of Phytochrome A-Regulated Gene Expression by Combined Analysis of Genomic Sequence and Microarray Data Plant Physiology, December 1, 2003; 133(4): 1605 - 1616. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. Inaba, Y. Nagano, T. Nagasaki, and Y. Sasaki Distinct Localization of Two Closely Related Ypt3/Rab11 Proteins on the Trafficking Pathway in Higher Plants J. Biol. Chem., March 8, 2002; 277(11): 9183 - 9188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nagano, H. Furuhashi, T. Inaba, and Y. Sasaki A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences Nucleic Acids Res., October 15, 2001; 29(20): 4097 - 4105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Makino, A. Matsushika, M. Kojima, Y. Oda, and T. Mizuno Light Response of the Circadian Waves of the APRR1/TOC1 Quintet: When Does the Quintet Start Singing Rhythmically in Arabidopsis? Plant Cell Physiol., March 1, 2001; 42(3): 334 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Inaba, Y. Nagano, J. B. Reid, and Y. Sasaki DE1, a 12-Base Pair cis-Regulatory Element Sufficient to Confer Dark-inducible and Light Down-regulated Expression to a Minimal Promoter in Pea J. Biol. Chem., June 23, 2000; 275(26): 19723 - 19727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nagano, T. Inaba, H. Furuhashi, and Y. Sasaki Trihelix DNA-binding Protein with Specificities for Two Distinct cis-Elements. BOTH IMPORTANT FOR LIGHT DOWN-REGULATED AND DARK-INDUCIBLE GENE EXPRESSION IN HIGHER PLANTS J. Biol. Chem., June 15, 2001; 276(25): 22238 - 22243. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
4). c, Comparison of
reporter-gene expression in different parts of intact stem. PL1
construct was bombarded into the indicated parts, and the
reporter-enzyme activity was measured as described.
