Xylem-specific expression of wound-inducible rice peroxidase genes
in transgenic plants
Hiroyuki Ito
a,*, Susumu Hiraga
a,b, Hidehito Tsugawa
c, Hirokazu Matsui
a,
Mamoru Honma
a, Yoshiaki Otsuki
d, Taka Murakami
e, Yuko Ohashi
b,eaDepartment of Applied Bioscience,Graduate School of Agriculture,Hokkaido Uni6ersity,Sapporo060-8589, Japan bCore Research for E6olutional Science and Technology (CREST),Chiyoda-ku,Tokyo 101-0062, Japan
cAomori Green BioCenter,Aomori 030-0142, Japan
dDepartment of Tea Agronomy,National Research Institute of Vegetables,Ornamental Plants and Tea,Kanaya 2769, Shizuoka 428-8501, Japan
eDepartment of Molecular Genetics,National Institute of Agrobiological Resources,Tsukuba,Ibaraki 305-8602, Japan
Received 6 September 1999; received in revised form 17 January 2000; accepted 19 January 2000
Abstract
A peroxidase gene, poxA, was isolated from a rice (Oryza sati6aL.) genomic library. The gene consists of four exons whose combined sequences were identical to that of the prxRPA mRNA whose levels were dramatically stimulated by wounding as well as by treatment of rice shoots with ethephon or UV irradiation [H. Ito, F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A.B. Caplan, R.L. Rodriguez, Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sati6aL.) shoots, Plant Cell Rep. 13 (1994) 361 – 366]. The temporal and spatial expression properties of thepoxAgene promoter as well as that from a second related peroxidase gene,poxN, were analyzed in transgenic tobacco and rice plants using the uidA gene as a reporter. In transgenic tobacco, UV- and wound-responsive
cis-elements were located within 144 bp from the translational start codon of thepoxAgene. ThepoxNpromoter, however, was inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimericuidAgenes containing 2.2 kb of thepoxApromoter or 1.4 kb ofpoxNpromoter were active in transgenic rice plants. Both peroxidase promoters directed GUS activities in a spatial and tissue specific manner coincident with the expression patterns exhibited by their mRNAs. Histochemical analysis of transgenic rice plants showed that both peroxidase genes are expressed in the vascular bundles of the shoot apex and lamina joint, and in xylem-parenchyma cells of the leaf blade and sheath. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Peroxidase; Rice (Oryza sati6aL.); Wound; Transgenic plants; Tissue-specific expression
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1. Introduction
Plant peroxidases (EC 1.11.1.7; donor:hydrogen peroxide oxidoreductase) are involved in several different physiological functions including wound healing [3,4], biosynthesis of cell walls [5 – 7], growth regulation [8], and senescence [9]. In view of their diverse roles, plants contain numerous peroxidase isoforms that are encoded by different
genes that comprise a complex multigene family. For example, rice shoots contain up to 25 or more enzyme isoforms as viewed by histochemical en-zyme analysis of protein extracts resolved by elec-trophoresis on native polyacrylamide gels [10]. Despite their importance in plant growth and de-velopment there have been very few reports on the purification and properties of peroxidase isoforms from rice plants and fewer attempts to assign a function for specific isoforms [10 – 12]. Because of recent plant genome project activities, many per-oxidase gene sequences or homolog sequences
* Corresponding author. Tel.: +81-11-7062500; fax: + 81-11-7063635.
E-mail address:[email protected] (H. Ito)
have been identified particularly in rice and Ara-bidopsis [13 – 19]. The primary sequences of the coded protein share about 40 – 50% homology and contain several highly conserved regions (60 – 90%) including two conserved histidine residues
pre-dicted to play a role in acid/base catalysis and to
serve as the fifth ligand of the heme prosthetic group [20].
The expression of the peroxidase genes, as well as pathogenesis-related protein genes [21], have been shown to be activated by a variety of envi-ronmental stimuli such as wounding [22], ethylene [23] and pathogen infection [12,24,25]. Few studies have been conducted to identify the regulatory
cis-elements present in the peroxidase promoters.
Mohan et al. [22] constructed chimeric uidA
fu-sions driven by the 5% flanking regions of the
tomato anionic tap1 and tap2 peroxidase genes
and showed that wound-induced GUS expression in transgenic plants. Recently, Klotz et al. [26] also prepared a chimeric gene composed of the pro-moter for the principal peroxidase gene found in
tobacco and theuidAgene, analyzed histochemical
GUS activity in transgenic tobacco plants, and suggested that the peroxidase is important in plant
growth and development rather than in
lignification.
Some cell wall-associated peroxidases catalyze the cross-linking and polymerization of phenolic polymers; it is one of key enzymes involved in lignin synthesis via phenylpropanoid pathway [27]. We believe characterization of the gene structure and the mode of gene expression of wound-in-ducible peroxidase isozymes is important not only to understand their biological functions involved in plant defence responses but to elucidate a part of signal transduction between wounding and gene expression.
In order to understand the function of peroxi-dases in plant growth and development, we iso-lated and characterized two peroxidase cDNA clones prxRPA and prxRPN [1]. A genomic clone
for prxRPN, poxN, was isolated and analyzed at
the structural level [2]. Here, we report the isola-tion and characterizaisola-tion of a second rice
peroxi-dase gene, poxA, which corresponds to the
prxRPA cDNA. We show that expression of the
poxA and poxN promoter-uidA fusion genes are
active in transgenic rice in a spatial and temporal pattern identical to that observed for their
respec-tive mRNAs. In contrast, only thepoxA promoter
is active in transgenic tobacco, indicating that one or more transcriptional regulatory elements of the
poxN promoter are not conserved between dicot
and monocot species.
2. Materials and methods
2.1. Plant materials
Rice (Oryza sati6a L. cv. Nipponbare) was
grown as described previously [1]. Rice suspen-sion-cultured cells were raised from seeds and
maintained in AA medium [28]. Tobacco (Nico
-tiana tabacumcv. Samsun NN) was grown at 25°C in a temperature-controlled greenhouse with a photoperiod of 16 h of light and 8 h darkness.
2.2. Isolation of a genomic clone
Genomic DNA was isolated from rice shoots, 21 days after germination, by the method of Mur-ray and Thompson [29]. The DNA was partially
digested with Sau3A and fragments (10 – 20 kb in
size) were recovered and inserted into the BamHI
site of lambda EMBL3 (Stratagene Cloning Sys-tems, La Jolla, CA, USA). The recombinant DNAs were packaged into bacteriophage particles (GIGA pack Gold-II; Stratagene) and grown on Escherichia coli LE392 (P2). The genomic library
was screened with a 32P-labeled prxRPA cDNA
using a BcaBEST labeling kit (Takara Shuzo,
Ky-oto, Japan) and [a-32P]dCTP (Amersham
Interna-tional, Buckinghamshire, UK).
2.3. Nucleotide sequencing
DNA was sequenced by the dideoxy chain-ter-mination method [30] using 7-deaza-dGTP instead of dGTP. The experimental details have been de-scribed previously [1]. The nucleotide sequences of
the poxA and poxN gene fragments appear in the
DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D84400 and D49551, respectively.
2.4. Primer extension
-GTAT-
TAAGGCACAGTACAAGAACAGAGCATAC-3%, for the poxA gene, and a 30-mer synthetic
oligonucleotide, 5%
-GAACGACGATATTGCA-GAGGAAACTCAGGC-3%, for the poxN gene
were end-labeled with [g-32P]ATP (Amersham
In-ternational) and a Megalabel kit (Takara Shuzo). The labeled primers were allowed to anneal
overnight at 30°C to 50 mg of total RNA
iso-lated from wounded shoots or healthy roots of
21-day-old rice seedlings. The shoots were
wounded by rubbing with sea sand and har-vested 2 days later. The extension reaction using reverse transcriptase RAV-2 (Takara Shuzo) was carried out at 42°C for 1.5 h. The products were analyzed on a 6% polyacrylamide sequencing gel containing 8 M urea. The same primer was used to produce a sequencing ladder of complemen-tary DNA.
2.5. Construction of promoter-uidA fusion genes
Six DNA fragments of the poxA promoter
were prepared by restriction enzyme digestions,
as follows: (i) Aa fragment, a 2204-bp KpnI –
ScaI fragment (position −2197 to +7; position
+1 is the A base of the translation start codon);
(ii) Ab fragment, a 1629-bp NotI –ScaI fragment
(−1622 to +7); (iii) Ac fragment, a 1138-bp
EcoRI –ScaI fragment (−1131 to +7); (iv) Ad
fragment, a 628-bp HindIII –ScaI fragment (−
621 to +7); (v) Ae fragment, a 355-bp P6uII –
ScaI fragment (−348 to +7); and (vi) Af
fragment, a 151-bp SphI –ScaI fragment (−144
to +7). Likewise, five DNA fragments of the
poxN promoter were prepared: (i) Nb fragment,
a 1640-bp XbaI –NheI fragment (−1425 to +
36); (ii) Nc fragment, a 1094-bp FspI –NheI
frag-ment (−1059 to +36); (iii) Nd fragment, a
801-bp NheI fragment (−766 to +36); (iv) Ne
fragment, a 410-bp HindIII –NheI fragment (−
375 to +36); and (v) Nf fragment, a 196-bp
EcoRV –NheI fragment (−161 to +36). The
termini of each DNA fragment were filled-in if
necessary and inserted into the filled-in HindIII
and XbaI sites of the pBI101 vector (Clontech
Laboratories, Palo Alto, CA, USA) [31]. The
re-sulting plasmids, designated pAa/GUS, pAb/
GUS, pAc/GUS, pAd/GUS, pAe/GUS,
pAf/GUS, pNb/GUS, pNc/GUS, pNd/GUS,
pNe/GUS, and pNf/GUS, respectively, were
grown in E. coli HB101 cells.
2.6. Transformation of tobacco plants
Plasmid DNA was introduced toAgrobacterium
tumefaciens LBA 4404 by electroporation [32].
Tobacco plants were co-cultivated with Agro
-bacterium by the leaf disc-infection method [33,34] and transformants were selected on Murashige and Skoog’s medium [35] supplemented with 100
mg/ml kanamycin and 250 mg/ml carbenicillin.
Regenerated plants were analyzed for the
integration of the promoter-uidAfusion genes into
the plant genome by the polymerase chain reaction (PCR). A sense primer sequence from the GUS
coding region (5%
-CTGCAGCGCTCACACCGA-TACC-3%) and an antisense primer sequence from
the gene for nopaline synthase terminator
(5%- ACAGGATTCAATCTTAAGAAACTTT - 3%)
were used for PCR. Primary transgenic plants and
progeny were referred to as T1 and T2
transformants, respectively.
2.7. Transformation of rice
Rice protoplasts were prepared from suspen-sion-cultured cells according to the method of
Otsuki [36]. Protoplast cells (1×106/mL) were
electroporated in a continuous flow electro-trans-fector (CET-100; JASCO, Tokyo, Japan) with 2
mg of plasmid containing the poxA or poxN
pro-moter-uidA gene and 0.4 mg of pUC19HPT
plas-mid (kindly provided by Dr A. Kato) that
included the CaMV35S promoter and
hy-gromycin phosphotransferase gene as described [37]. Formation of hygromycin-resistant calli from transformed protoplasts and regenerated plants from the calli were carried out as de-scribed [36]. Transformants were initially selected by checking roots for GUS activity. Regenerated plants were analyzed for the integration of the introduced construct into the genome by PCR. In addition, the integration of genes was also confirmed by Southern blot hybridization. Five microgrammes of total DNA from transgenic
rice plants containing the introduced the AA/
GUS or NB/GUS fusion gene was digested with
EcoRI or XbaI and SacI, respectively,
fractiona-tioned by electrophoresis on a 0.7% agarose gel, and blotted onto nylon membrane (Amersham
International plc). A 0.5-kb HincII fragment
from the uidA gene from the pBI101 plasmid
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 mE/s/m2)/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
(c600; 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 (c600). 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% H2O2for 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 mg/mL
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
3×105 recombinant phages was screened with
32P-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, designatedlpoxA, was selected for further
study.
Based on hybridization studies thepoxAgene was
located on a 4237-bpKpnI/HindIII 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
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,
thepoxAand poxNgenes 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 thepoxAgene was a cytosine base
located 38 bp upstream of the AUG codon. Under the same experimental conditions, the
transcrip-tion initiatranscrip-tion 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
Table 1
Sequences of putativecis-elements found in thepoxAandpoxN promoters
Gene Sequence (positionb)
Element Motif or core sequencea Reference
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)
aW and M in the motif or core sequences indicate A or T and C or A, respectively.
bThe 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
theuidAgene 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 thepoxA
gene showed significantly elevated levels of GUS activity after UV treatment. An identical
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 theuidAgene to generate the
plasmids, pAa/GUS and pNb/GUS, 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 Aa/GUS gene and 4 of
14 individuals containing the Nb/GUS 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, Aa/GUS or Nb/GUS, 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 thepoxApromoter
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 Aa/GUS and Nb/GUS
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 Aa/GUS 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 andpoxN 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.
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 withEcoRI orXbaI/SacI, 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
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
4.1. Gene Structure
In a previous study [2] we determined the
struc-ture of thepoxN gene which encodes a rice
perox-idase which is distinct from the enzyme encoded
by poxA. Comparison of the poxA and poxN
genes indicates that they have a similar structure of four exons and three introns. More than 50 genes for plant peroxidases have been reported in the various DNA sequence databases (GenBank, EMBL and DDBJ). About 60% of these
peroxi-dase genes, including the poxA and poxN genes,
contain an initial transcription unit of four exons interrupted by three introns. Each of the three introns is located at identical positions within the exon sequences that code for the highly conserved primary sequences of the protein. This gene ar-rangement supports the view that these genes evolved from a common ancestor.
Despite the similarity in overall gene structure,
the poxA and poxN genes encode peroxidase
en-zymes that are likely to play different roles in rice growth and development. The introns and, more importantly, promoter sequences of these genes differ considerably at the DNA sequence level. This sequence divergence in the promoter regions of these genes is likely to reflect differences in their
transcription levels, and in their temporal and spatial patterns of gene expression and, in turn, differences in their function during rice growth and development.
Our previous study [1] showed that both the
poxA andpoxNtranscripts are induced by
wound-ing, treatment with ethephon, and UV-C irradia-tion in rice plants. Consistent with the inducirradia-tion by ethephon, both promoters contain GCC box motives [2]. The GCC box is an ethylene-responsi-ble element and has been found in the promoters of ethylene-inducible PR family of genes (basic PR
genes containing chitinases, and b-1,3-glucanases)
[42,43], but not in ethylene-responsive genes in-volved in ripening and senescence [47,48]. The
presence of this cis-regulatory element and
induc-tion of gene expression by wounding supports the
possibility that the poxA and poxN gene products
belong to the PR family which are closely associ-ated with the ethylene-induced self-defence re-sponse [21,49].
4.2. The acti6ities of the poxA and poxN promoters in transgenic tobacco plants
Various deleted forms of the poxA and poxN
promoters were fused to the uidA gene and the
state levels of both transcripts were dramatically stimulated by wounding, by treatment of ethephon and by exposure to UV irradiation [1]. However, except for a small increase in promoter activity that was reproducibly observed in AF plants (Fig.
2), none of the other transgenic plants showed an increase in GUS activity by treatment with ethep-hon or by wounding in tobacco (Fig. 2). These results suggest that there is an UV responsible
element located within the −106 bp of the
scription start site of the poxA gene and one or more negative regulatory elements, located
be-tween −310 and −106 bp, which suppress the
downstream promoter activity. Two silencer-like sequences similar to that observed in the bean chalcone synthase promoter [46] are located just
upstream of the Af region, GGTTGGAA at −
308 bp and GGTTAGCAC at −265 bp.
Much different poxA and poxN gene expression
patterns to these abiotic stress conditions were seen in transgenic rice. The promoters of both genes responded to wounding and treatment with ethephon or by UV irradiation. These differences in expression patterns between rice and tobacco plants are likely to reflect differences in the tran-scriptional machinery between the monocot rice and dicot tobacco plants. There are reports that monocot genes are sometimes expressed only at low levels in a heterologous dicot plant [50,51]. Additionally, our results indicate the possibility that the two putative silencer elements found in the Ae sequences upstream of the Af region are only effective in tobacco plants but not in rice plants.
4.3. Expression of poxA and poxN promoters in transgenic rice plants
Unlike the situation in transgenic tobacco where the rice peroxidase promoters, except for one in-stance, are inactive (Fig. 2), these promoters direct the same expression patterns as the endogenous peroxidase genes in transgenic rice plants (Fig. 4). Since preparation of the tissue for histochemical staining for GUS activity treatments results in tissue wounding, it is difficult to observe xylem
parenchyma specific expression of the poxA or
poxN gene without any stress treatment. The
ex-pression and its induction of GUS activity (Fig. 4) in transgenic rice plants and northern analyses [1], however, support the view that these genes are expressed at low levels in leaf blade or sheath without stress. Treatment with ethephon or UV-C irradiation and wounding induces their expression. It should be pointed out that ethephon treatment may be not be equivalent to ethylene because ethephon degradation leads not only to the gener-ation of ethylene but also the accumulgener-ation of acids [52].
In transgenic rice plants, AA1 – T2, GUS activ-ity was observed in vascular bundles of lamina
joints and palea from unstressed plants (Fig. 5J,
K, L, and M), suggesting the poxA gene
expres-sion is specifically regulated in a spatial and devel-opmental manner. GUS was also expressed in lamina joints of NB1 – T2 plants but not in the palea (data not shown). Interestingly, lamina joints have been used for a flexuous test of gravit-ropism or phototgravit-ropism via auxin [53]. The ex-pression of these genes in this tissue supports the suggested involvement of peroxidases with auxin metabolism.
Many studies have demonstrated that plant per-oxidases are involved in IAA metabolism [54,55] and the general scheme for IAA oxidation has also been proposed [56]. Recently, Savitsky et al. [57] showed that plant peroxidases share common sub-domains including the catalytic center with plant
auxin-binding proteins. It is unclear if the poxA
peroxidase binds IAA and catalyzes their oxida-tion, but the gene expression in lamina joints suggests such a possible role. Although there are eight vascular bundles in rice caryopsis, GUS was observed in only one vascular bundle of the palea,
suggesting that the poxA gene is expressed in a
strict spatial manner.
BothpoxA andpoxN promoters contain several
Box P-like sequences (Table 1). The Box P element has been identified in the genes that code for enzymes important in the biosynthesis of lignin, lignans and phenylpropanoids including pheny-lalanine ammonia-lyases, 4-coumarate:CoA
lig-ases, and cinnamyl alcohol dehydrogenases
[44,46]. It has been reported that a parsley nuclear protein, BPF-1, binds to the Box P motif and plays an important role in plant defence responses
[44]. Hauff et al. [58] identified the cis-acting
re-gion that is critical for expression of parsley gene for a 4-coumarate:CoA ligase in vascular tissues.
This cis region contains a negative regulatory
ele-ment that suppresses expression in the phloem and a positive element that promotes expression in the
xylem. The product of the maizePgene, amyb
expres-sion of the poxA and poxN genes. Because genes involved in lignin and lignan biosynthesis are ex-pressed specifically in the xylem or vascular tissue
[45,58,61,62], the localization of poxA and poxN
encoded peroxidase to these tissues support their involvement in lignin and lignan biosynthesis. Fu-ture efforts will be directed at elucidating the specific function of these enzymes in these con-ducting tissues.
Acknowledgements
We are grateful to Dr A. Kato for providing the plasmid pUC19HPT. The authors thank Y. Gotoh for her excellent technical assistance. This work was supported in part by the fund for the En-hancement of Centers of Excellence (COE), the Special Coordination Funds for the Promotion of Science and Technology in Japan, and by Grants-in-Aid for Scientific Research (nos. 06760065 and 07760069) from the Ministry of Education, Sci-ence and Culture, Japan.
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