Plant Science 149 1999 85 – 94
Isolation and characterization of a cDNA clone encoding asparagine synthetase from root nodules of Elaeagnus umbellata
Ho Bang Kim, Sang Ho Lee, Chung Sun An
Department of Biology, Seoul National Uni6ersity, Seoul
151
-
742
, South Korea Received 15 September 1998; received in revised form 24 June 1999; accepted 28 June 1999
Abstract
A cDNA clone encoding asparagine synthetase AS was isolated from a root nodule cDNA library of Elaeagnus umbellata by competitive hybridization. The clone, pEuNOD-AS1, coded for 585 amino acid residues with molecular weight of 65.8 kDa and
pI value of 6.12. Expression of AS was highly enhanced in the root nodule, and its expression pattern during nodule development was very similar to that of nifH, showing highest level 6 – 8 weeks after inoculation and decreased thereafter. In situ hybridization
result showed AS transcripts were strongly detected in the infected cells of fixation zone, where nifH transcripts were also detected. These results suggest its expression may be under metabolic control rather than developmental control of the root nodule.
Genomic Southern hybridization revealed the presence of at least two AS genes in the genome of E. umbellata. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Asparagine synthetase; cDNA; Elaeagnus umbellata; In situ hybridization; Nodule-enhanced expression; Root nodule www.elsevier.comlocateplantsci
1. Introduction
Actinorhizal root nodules are nitrogen-fixing symbioses involving the actinomycete Frankia and
roots of dicotyledonous plants belonging to eight different families and 25 genera. Most are capable
of high rates of nitrogen fixation comparable to those found in legumes [1]. Actinorhizal root nod-
ules resemble lateral roots with a central vascular system and originate from pericycle, while legumi-
nous root nodules resemble shoots with peripheral vascular systems and originate from cortex [2].
Due to the presence of apical meristem activity, its nodule lobes show indeterminate growth pattern
and thus consist of four different zones, which are meristem zone, prefixation zone, fixation zone and
senescence zone [3]. For these reasons, the acti- norhizal root nodules may be useful system to
study many aspects of plant development. Ammonia fixed by dinitrogenase in root nodule
should be transported to other parts of the plant through xylem after assimilation into amino acids.
The type of amino acids to be transported through xylem are different according to plant species,
developmental stage or environmental conditions. Generally symbiotic legumes and actinorhizal
plants can be divided into amide transporters temperate legumes and actinorhizal plants such as
Datisca, Elaeagnus, Myrica, etc. and the ureide transporters tropical legumes and actinorhizal
plants such as Alnus, etc. [4]. Until now, studies of molecular aspects of enzymes related with am-
monia assimilation in root nodules of actinorhizal plants were concentrated into Alnus, a ureide
transporter. Recently, cDNA clones encoding glu- tamine synthetase, a component of GSGOGAT
enzyme complex, and acetylornithine transami- nase, an enzyme related with citrulline biosynthe-
sis, from the root nodule of Alnus glutinosa were isolated and their expression patterns were charac-
terized [5].
Corresponding author. Tel.: + 82-2-880-6678; fax: + 82-2-872- 6881.
E-mail address
:
ancsplaza.snu.ac.kr C.S. An 0168-945299 - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 2 4 - 7
Asparagine Asn is a major amino acid form transporting nitrogen in plants faced with condi-
tions of excess ammonia for example, germina- tion, growth on fertilizers and nitrogen fixation
and limitation of carbon source [6,7]. Asn is an ideal amino acid form for transport of reduced
nitrogen, because of its higher N:C ratio 2:4 than glutamine 2:5 and its stability [8]. The synthesis
of Asn is mediated by asparagine synthetase AS, EC 6.3.5.4, which catalyzes the ATP-dependent
transamination reaction transferring the amide group of glutamine or ammonia to aspartate,
resulting in the formation of glutamate and Asn while hydrolyzing ATP to AMP and PPi;
L-Asp + L-Gln + ATP L-Asn + L-Glu + AMP + PPi.
Two different types of AS have been described in E. coli and yeast; a Gln-dependent form and an
ammonia-dependent form [9,10]. Although all plant ASs studied up to date appear to be Gln-de-
pendent form, AS in maize roots can use ammonia as a substrate effectively under the condition of
excess ammonia [11]. Biochemical study of AS has been hampered by its extremely low stability, con-
taminating asparaginase activity and specific non- protein inhibitors [12]. Molecular and genetic
studies have been used to circumvent the difficulty of biochemical study. Significant progress has been
made in understanding expression and regulation of AS through the isolation and characterization
of cDNA andor genomic clones from non-nodu- lating plants such as asparagus [13], Arabidopsis
[14], broccoli [15] and maize [16], and especially from legume plants such as pea [17], Lotus japoni-
cus [18], alfalfa [19], soybean [20] and broad bean [21]. Nothing, however, has been known on the
expression and regulation of AS from any acti- norhizal plants.
In this paper, a cDNA clone encoding AS was isolated from the root nodule cDNA library of E.
umbellata, a amide transporter [4], by competitive hybridization and its molecular biological aspects
were characterized. Expression pattern of AS in different tissues was investigated by Northern hy-
bridization. Expression pattern of AS during nod- ule development and distribution of AS transcripts
in the root nodule were analyzed by RT-PCR Reverse
Transcriptase-mediated Polymerase
Chain Reaction and in situ hybridization, respec- tively. This is a first report for isolation and
characterization of cDNA clone encoding enzyme related with ammonia assimilation in the root
nodule of amide transporting actinorhizal plants.
2. Materials and methods
