Plant Science 155 2000 85 – 100
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,e
a
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido Uni6ersity, Sapporo
060
-
8589
, Japan
b
Core Research for E6olutional Science and Technology CREST
, Chiyoda-ku, Tokyo
101
-
0062
, Japan
c
Aomori Green BioCenter, Aomori
030
-
0142
, Japan
d
Department of Tea Agronomy, National Research Institute of Vegetables, Ornamental Plants and Tea, Kanaya
2769
, Shizuoka
428
-
8501
, Japan
e
Department 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 sati6a L. 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 sati6a L. shoots, Plant Cell Rep. 13 1994 361 – 366]. The temporal and spatial expression properties of the poxA gene 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 the poxA gene. The poxN promoter, however, was
inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimeric uidA genes containing 2.2 kb of the poxA promoter or 1.4 kb of poxN promoter 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 sati6a L.; Wound; Transgenic plants; Tissue-specific expression www.elsevier.comlocateplantsci
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
:
otihchem.agr.hokudai.ac.jp H. Ito 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 0 9 - 0
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 acidbase 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 tap
1
and tap
2
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 the uidA gene, 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 characterization 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 the poxA 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
