labeling kit Takara Shuzo, and used for the hybridization.
2
.
8
. Exposure of transgenic plants to 6arious stresses
Leaf discs ca. 7 mm in diameter containing a small vein from various transgenic tobacco plants
were incubated in either sterile water control treat- ment, 1 mM 2-chloroethylphosphonic acid ethep-
hon treatment, or 0.4 mM abscisic acid ABA treatment for 48 h. Leaf discs were also exposed to
UV-C light for 30 min 15 min per side at a distance of 30 cm from an UV lamp sterilization lamp
GL-15; Toshiba, Tokyo, Japan; before incubating in water for 48 h UV irradiation treatment. Leaf
discs from healthy untransformed tobacco plants were also treated as mentioned above and were used
as controls. The 48 h incubation period consisted of two diurnal cycles of 16 h light 45 mEsm
2
8 h dark cycle at 25°C.
Detached leaf blades from several transgenic rice plants were cut to small pieces about 3 cm in length
and incubated in sterile water, in ethephon, or in ABA, or treated with an UV-lamp, as described for
the transgenic tobacco plants. Leaf blades were also wounded by rubbing the leaf with carborundum
c 600; Nacalai Tesque, Kyoto, Japan before incubating in water for 48 h. In analysis of trans-
genic rice plants, the 48 h incubation was performed at 25°C in dark.
2
.
9
. Measurement of GUS acti6ity GUS activity was assayed by the method of
Kosugi et al. [38]. Leaf discs from transgenic tobacco plants were homogenized in lysis buffer 50
mM sodium phosphate pH 6.8, 10 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 Triton X-100, and
0.1 sarcosyl in a Eppendorf tube with a glass rod and carborundum c 600. Leaf blades from trans-
genic rice plants were homogenized in lysis buffer with carborundum by a mortar and pestle. The
homogenate was centrifuged at 10 000 × g for 15 min and the supernatant was assayed for GUS
enzyme in the presence of 5 methanol. Fluores- cence levels were determined using a Shimazu
RF-540
spectrofluorometer Shimazu,
Kyoto, Japan. A unit of GUS activity is defined as 1 pmol
of 4-methyl-umbelliferone 4-MU produced per minute per milligram of protein. Protein concentra-
tions were determined by the Bradford method [39] using a commercial kit BioRad Laboratories, Rich-
mond, CA, USA and bovine serum albumin as the standard.
2
.
10
. Histochemical analysis Tissue sections from transgenic rice plants were
made to 80 – 100 mm with a microslicer model DTK-1000, Dosaka, Kyoto, Japan. Histochemical
staining for GUS activity was carried out in 50 mM sodium phosphate buffer pH 7.0 containing 1 mM
5-bromo-4-chloro-3-indolyl glucuronide X-Gluc and 5 methanol as described [34,38]. For peroxi-
dase activity staining, tissue sections were incubated in McIlvaine’s buffer pH 5.0 containing 0.03
4-chloro-1-naphtol and 0.001 H
2
O
2
for 30 min at room temperature. The reaction was stopped by the
addition of 0.2 N sodium carbonate. Lignification was detected by staining with 8.3 mgmL phloroglu-
cin in 50 ethanol and 0.3 N HCl.
3. Results
3
.
1
. Isolation and characterization of the poxA gene A genomic library consisting of approximately
3 × 10
5
recombinant phages was screened with
32
P-labeled prxRPA cDNA [1] as a probe. Four positive clones were obtained after the second
screening. These clones were plaque-purified and their DNAs purified for analysis of the inserted
DNA fragments. As restriction physical mapping analysis indicated that all of the phage DNAs had
the same restriction pattern, only one of the four DNAs, designated lpoxA, was selected for further
study.
Based on hybridization studies the poxA gene was located on a 4237-bp KpnIHindIII fragment which
was sequenced in both directions. Comparison of the genomic sequence with the cDNA revealed that
the gene consisted of three introns and four exons Fig. 1. The nucleotide sequences of the exons were
identical to that of the cDNA. All introns contained a high percentage of A + T residues 64 – 70, a
feature characteristic of higher plants genes [40]. In contrast, the four exons had a lower A + T 40 –
52 compositions. The 5 and 3 ends of each intron had the consensus border sequences GT and
AG, respectively [41].
The poxA gene structure is similar to the previ- ously isolated poxN gene [2] in containing three
introns and four exons Fig. 1. Its deduced amino acid sequence shares significant homology 63
identity and 85 similarity to the product coded by the poxN gene. Despite this similarity in gen-
eral structural organization and coding regions, the poxA and poxN genes contained unique intron
regions which differ in length and in DNA se- quence. Moreover, the 5-promoter and 3-untrans-
lated regions of these genes shared very little homology.
Since our previous results had shown that mR- NAs corresponding to prxRPA and prxRPN cD-
NAs accumulated at low levels in healthy rice shoots, we isolated total RNA from wound-
treated shoots for a primer extension experiment. We also used total RNA from healthy rice roots,
in which the transcripts accumulate to high levels. In both instances, the determined transcription
initiation site of the poxA gene was a cytosine base located 38 bp upstream of the AUG codon. Under
the same experimental conditions, the transcrip- tion initiation site of the poxN gene was not
detected.
3
.
2
. Characterization of the poxA and poxN promoter regions
Putative TATA boxes in the poxA and poxN genes are located at 70 and 91 bp, respectively,
upstream from the translational start. Because both of these peroxidase genes are wound-in-
ducible, the poxA and poxN promoter regions, were scanned for sequence motifs suggested to be
cis-regulatory elements in plant defence-related genes Table 1. One sequence motif, 5-AGC-
CGCC-3 a GCC box, is conserved in the pro- moter regions of the pathogenesis-related proteins,
b-1,3-glucanase and chitinase and is reported to be an essential sequence for ethylene responsiveness
[42,43]. The poxA and poxN promoters contain this sequence motif at positions − 141 and − 222,
respectively, from the translation start. Within both promoter regions, there are also several ho-
mologs of the Box P motif found in several phenylpropanoid
gene promoters,
particularly those of phenylalanine ammonia-lyase and 4-cou-
marate:CoA ligase genes. Sequences similar to the myb-related protein binding site encoded by the
maize P gene [44,45] are also seen. Several se-
Fig. 1. Restriction maps of poxA gene in lpoxA clone and poxN genes. The shaded boxes denote exon regions for each gene. The number below each of the exons and introns indicate the length in bp. The AUG initiation codon was used as the starting point
for the first exon indicated by asterisks for each gene.
Table 1 Sequences of putative cis-elements found in the poxA and poxN promoters
Gene Sequence position
b
Element Reference
Motif or core sequence
a
AGCCGCC GCC box
poxA AGCCGCC −141
[42,43] poxN
AGCCGCC −222 Box P
ACMWAMC poxA
CACACCGACCA −2026 [44,45]
ACTAAACCAAC −1029 CACCCACTACCAC −338
poxN AACCGACCTAGCC −1253
CACCAACCA −1075 SBF
GGTTAAWWW poxA
GGTTCTATT −877 [46]
CGTTAAAAT −367 GGTTGGAA −346
poxN GGTTTATA −938
GTTTATAAT −880 GATTAAGTT −541
CCWACC Maize P
poxA CCTATC −115
[59] poxN
CCAACC −1073
a
W and M in the motif or core sequences indicate A or T and C or A, respectively.
b
The positions are given by the number corresponding to the 5 nucleotide in the motif from the translational start codon.
quences homologous to the silencer consensus SBF sequence, GGTTAAWWW W is A or T,
observed in the bean chalcone synthase promoter [46] are present in both peroxidase 5 flanking
regions.
3
.
3
. Promoter acti6ity in transgenic tobacco plants To investigate the functional properties of the
poxA and poxN promoters using a transgenic ap- proach, a series of 5-promoter deletions fused to
the uidA gene were constructed as shown in Fig. 2. Each of the DNA constructs was introduced into
tobacco leaf cells by Agrobacterium infection and regenerated kanamycin resistant plants obtained.
The presence of the introduced genes was verified by PCR analysis of genomic DNA and stress-in-
duced GUS activity assayed in leaf discs of the T1 transgenic plants.
The series of transgenic tobacco plants contain- ing the various 5 promoter deletions of either the
poxA and poxN promoters were treated with ei- ther ethephon, ABA, or UV light Fig. 2. Leaf
discs from all of the transgenic plants incubated in water alone for 48 h contained only low levels of
GUS activity. Ethephon or ABA treatment had no effect on the level of GUS activity from plants
containing any of the DNA promoter constructs when compared to the control water treatment.
Likewise, all of the plants, except for one, were unaffected by UV light treatment. AF, which con-
tained the smallest promoter fragment of the poxA gene showed significantly elevated levels of GUS
activity after UV treatment. An identical UV-re-
Fig. 2. Construction of promoter-uidA gene fusions and induction of GUS activity in transgenic tobacco leaves. A The fragments Aa to Af and Nb to Nf were inserted into the pBI101 vector to construct the various plant expression plasmids. The brackets
indicate the names of each plasmid and the corresponding transgenic plant. The numbers in the parentheses show the positions from the translational initiation codon. RB and LB, right and left borders, respectively, of T-DNA; Pnos and Tnos, promoter and
terminator, respectively, of nopaline synthase gene; KanR, kanamycin-resistance gene. B DNA and amino acid sequences depicting the fusion of the GUS gene to the poxA or poxN promoters. Lower-case letters indicate the sequence of the vector. C
GUS activities observed in transgenic tobacco leaf discs after application of various stresses. Leaf discs from control plants and each transgenic tobacco plant were treated with 0.1 M ethephon, 0.4 mM ABA, or UV irradiation. After incubation for 48 h in
darkness at 25°C, GUS activities of leaf discs were measured. GUS activities are represented as plot of results from 6 to 14 different transgenic plants. The average is indicated as a bar. C indicates untransformed tobacco plants. Ab through Af and Nb
through Nf indicate transgenic tobacco plants that harbored plasmids pAbGUS through pAfGUS and pNbGUS through pNfGUS, respectively.
Fig. 2.
sponse pattern of the AF plants was observed in T2 transgenic plants. These results indicate that
the poxA gene contains a UV-responsive cis ele- ment located within − 144 bp of translation start
codon that functions in tobacco plants and that a strong silencing sequence exists between − 348
and − 144 bp. No induction of GUS activity was detected for any transgenic tobacco plants con-
taining the poxN promoter.
3
.
4
. Promoter acti6ity in transgenic rice plants The 2.2-kb poxA and 1.5-kb poxN promoter
regions were fused to the uidA gene to generate the plasmids, pAaGUS and pNbGUS, respectively
Fig. 2. Both plasmids were introduced into rice protoplasts by electroporation and cultured for the
production of transgenic plants. Since both genes were constitutively expressed in healthy roots,
GUS activity in the roots of juvenile regenerated plants was assayed histochemically. Nineteen of 24
individuals containing the AaGUS gene and 4 of 14 individuals containing the NbGUS gene exhib-
ited GUS activity data not shown. These trans- formants were named AAn – T1 or NBn – T1 plant
AA and NB refer to the introduced chimeric gene, AaGUS or NbGUS, and n denotes an
independent line, while T1 means the primary transformed plants. Four lines of AAn plants
AA1 – AA4 and three lines of NBn plants NB1 – NB3 were selected for further analysis because of
their high GUS activities in roots. A third series of plants containing the 0.6-kb of the poxA promoter
region fused to the GUS gene were obtained as well. No GUS activity, however, was found in the
roots from more than independent 40 transfor- mants indicating that this smaller promoter frag-
ment lacked one or more cis-regulatory elements for root expression.
To confirm whether the AaGUS and NbGUS chimeric genes were integrated into the plant
genome, Southern blot was performed on DNAs isolated from AA1 – AA4 and NB1 – NB3 plants
Fig. 3. The coding region 0.5 kb HincII frag- ment of the uidA gene was used as a probe after
digestion of genomic DNAs from AA plants with EcoRI or from NB plants with XbaI and SacI.
The results indicate that the AaGUS and Nb GUS genes were integrated in the genomes of the
transgenic plants. In addition to the intact gene insertions, rearranged gene copies were also
detected. Healthy rice shoots when subjected to wound-
ing, UV irradiation, or treatment of ethephon showed increase levels of poxA and poxN tran-
scripts [1]. We examined whether the hybrid trans- genes behaved similarly to these abiotic stresses.
Fig. 4 shows the changes of GUS activities in leaf blades of the transgenic plants after the various
stress treatments. A slight increase in GUS activity was observed in control leaf blades which were
incubated in sterilized water for 48 h. When the isolated leaf blades were treated with ethephon,
UV irradiation, and wounding, a several-fold in- duction of GUS activity was observed. In contrast,
no significant increases in GUS activity over the water control was evident when the leaves were
treated with ABA. These results indicate that these poxA and poxN promoter fragments contain all of
the cis-elements required for faithful expression patterns as seen for the native genes.
3
.
5
. Localization of GUS acti6ity, lignin, and peroxidase acti6ity in transgenic rice plants
To determine the spatial expression patterns of the poxA and poxN genes, histochemical staining
of GUS activities was performed on various tis- sues of the transgenic rice plants. In AA1 – T1
plants, GUS activities were observed in the vascu- lar bundle and cylinder of the shoot apex Fig. 5A
and B, in the xylem parenchyma cells of the leaf blade, and in the root exodermis, endodermis and
percycle Fig. 5C and G. In NB1 – T1 transgenic plants, the same pattern of GUS activities as in
AA1 – T1 plants was observed in the shoot apex and roots data not shown. In addition, GUS
activity was detected in xylem parenchyma cells of leaf sheath Fig. 5H. In both plants, GUS expres-
sion was found in the large vascular bundle of not only the leaf blade but also in the leaf sheath. In
both AA1 – T1 and NB1 – T1 plants, GUS staining predominated in the leaf blade as compared to the
sheath.
Fig. 5F shows the distribution of peroxidase activity when visualized with 4-chloro-1-naphtol.
Peroxidase activity was detected around the large vascular bundle and mesophyll cells of the leaf
sheath in NB1 – T1 plants. The peroxidase activity was much stronger in mesophyll cells than the
vascular bundles. The same staining pattern of peroxidase activity was observed in leaf blade and
sheath of AA1 – T1 plants as well as in healthy rice
plants. GUS expression in xylem parenchyma cells coincided with or was very close to areas of lignifi-
cation Fig. 5D, E, and I. These observations suggest that both peroxidases may be involved in
lignification of the large vascular bundle. When young whole AA1 – T2 plants were
stained with X-Gluc without any stress treatments, GUS was detected in roots particularly in the stele
Fig. 5K as well as the vascular bundles of the lamina joint Fig. 5L and palea Fig. 5M.
Fig. 3. Southern hybridization of AAn and NBn transgenic rice plants. Total DNA from the AAn – T1 or NBn – T1 rice plants was digested with EcoRI or XbaISacI, respectively, and processed for Southern blotting using standard techniques. The lane
number denotes an independent transgenic line from AAn – T1 or NBn – T1 plants. Lanes C1 and C2 were applied. EcoRI and XbaISacI digested total DNA, respectively, from non-transgenic rice plants. The arrow indicates the band sizes that should be
detectable in restricted fragments from AAn – or NBn – T1 plants.
Fig. 4. Induction of GUS activity in transgenic rice plants. Leaf blades from each transgenic rice plant were treated with 0.4 mM ABA A, 0.1 M ethephon E, UV irradiation U, or wounding W. After incubation for 48 h at 25°C, GUS activities were
measured. 0 indicates the level of GUS activity of a healthy leaf blade. H depicts the level of GUS activity in leaf blades incubated in water mock treatment. GUS activities shown are an average of six leaf blades subjected to various treatments.
4. Discussion
