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
2
.
1
. Bacterial strain and plant material Frankia strain EuIK1, a symbiont of E. umbel-
lata root nodule, was used to nodulate E. umbel- lata seedlings. Culture methods for E. umbellata
seedlings and the Frankia strain were described previously [22]. RNAs for construction of nodule
cDNA library were isolated from root nodules at various developmental stages 6 months after inoc-
ulation. Uninoculated seedlings were used to iso- late RNAs of leaf and root. To study gene
expression level during nodule development, nod- ules of 4, 6, 8, and 10 weeks after inoculation were
harvested and stored at − 80°C until used. For in situ hybridization, nodules of 8 – 10 weeks after
inoculation were used.
2
.
2
. Isolation of DNA and RNA The method of Doyle and Doyle [25] for isola-
tion of genomic DNA was modified to isolate total RNA from leaves, roots and nodules of E. umbel-
lata. Plant tissues were ground in liquid nitrogen. Polyvinypolypyrrolidone PVPP was added dur-
ing grinding with liquid nitrogen to remove pheno- lic compounds. CTAB cetyltrimethyl ammonium
bromide extraction buffer 10 – 12 mlg tissue was added to the tissue powder. The homogenate was
incubated at 60°C for 10 min and extracted with phenol and chloroform. The supernatant was pre-
cipitated with cold IPA and washed with washing buffer [76 vv EtOH, 10 mM ammonium ac-
etate]. Dried pellet was dissolved with nuclease- free
water. Total
RNA was
differentially precipitated with lithium chloride from total nu-
cleic acids and treated with RNase-free DNase to remove genomic DNA. PolyA
+
RNA for nodule cDNA library construction was purified from total
RNA using
oligo dT
cellulose column
Boehringer Mannheim, Mannheim, Germany ac- cording to the methods of Ausubel et al. [24].
Genomic DNA was isolated from E. umbellata leaves according to Doyle and Doyle [25]. Plasmid
DNA was purified according to Sambrook et al. [26].
2
.
3
. Construction and screening of a cDNA library
cDNA was synthesized from 5 mg of polyA
+
nodule RNA using cDNA synthesis Kit Strata- gene, La Jolla, CA, USA according to manufac-
turer’s guide. The synthesized cDNA was inserted into ZAP express vector Stratagene and recombi-
nant phage DNA was in vitro packaged using Gigapack III Packaging Extract Stratagene ac-
cording to manufacturer’s guide. Phage DNAs from about 10 000 plaques per petri dish r = 4.5
cm were transferred to nylon membranes Amer- sham, Berckinghamshire, UK and cross-linked by
UV-treatment. Ten phage blots were competitively hybridized with nodule cDNA probe and an ex-
cess of total RNA from roots and leaves according to Mangiarotti et al. [27]. The cDNA probe was
synthesized from 2 to 5 mg polyA
+
nodule RNA.
2
.
4
. Cloning and sequence analysis Positive phage clones from primary and sec-
ondary competitive screening were changed into phagemid clones according to in vivo excision
protocol of manufacturer Stratagene. Phagemid DNA was deleted unidirectionally with exonucle-
ase III and S1 nuclease by using double-stranded Nested Deletion Kit Pharmacia Biotech, Uppsala,
Sweden based on the protocol of Henikoff [28]. The nucleotide sequences were determined by
dideoxynucleotide chain termination method [29] using Taq polymerase Promega, Madison, WI,
USA and T7 DNA polymerase USB, Cleveland, OH, USA. The sequences were analyzed using
PC-GENE Release 6.01; IntelliGenetics, Moun- tain View, CA, USA, DNASISPROSIS V6.01;
Hitachi Software Engineering, Tokyo, Japan and BLAST search program [30,31].
2
.
5
. DNA and RNA blot analysis For DNA analysis, genomic DNAs from leaves
of E. umbellata 10 mg digested with several re- striction enzymes EcoRI, HindIII and BglII
were electrophoresed on 0.8 agarose gel, and transferred to Hybond-N membrane Amersham
by capillary blotting method [26]. For RNA analy- sis, total RNAs from leaves, roots and nodules of
E. umbellata 10 mg were electrophoresed on a 1 glyoxal gel and transferred to Hybond-N mem-
brane Amersham by capillary blotting method [26]. The blots were hybridized overnight with
32
P-labelled AS probe under following condition; 6 × SSC,
5 × Denhardt’s solution,
0.5 SDS
Sodium Dodecyl Sulfate at 65. The hybridized blots were washed at 65°C with gradually decreas-
ing salt concentration to 0.5 × SSC, 0.1 SDS, and exposed to X-ray film.
2
.
6
. RT-PCR RT-PCR method was used to analyze expres-
sion pattern of AS gene in the course of nodule development. Two PCR primers [upper primer
position 1715 – 1737; 5-TTCTGGAAGGGCTG- CACTAGGAG-3, lower primer position 1913 –
1937; 5-TCCCCATCAGGCATAGAATCCAT- T-3] were designed to specifically amplify 3 UTR
untranslated region of AS cDNA clone. Total RNAs 1 mg from each developmental stage were
used as template for reverse transcription after RNase-free DNase Promega treatment. To assess
nitrogenase activity during nodule development, 618 bp between position 88 and position 705 of
nifH ORF encoding nitrogenase reductase [32] was amplified. Transcripts of PUB polyubiquitin
were also amplified as an indirect RT-PCR control using two primers specific to 3 UTR of PUB
cDNA clone [23]. PCR cycling conditions for AS nifH and PUB were 95°C for 5 min for initial
denaturation followed by 95°C for 1 min, 62°C 67°C for nifH for 1 min and 72°C for 1 min 30
cycles with 10 min final extension at 72°C. Am- plified PCR products were electrophoresed on
agarose gel, transferred to nylon membrane, and probed with inserts of AS, PUB and nifH clone.
The
hybridization signals
of the
blot were
quantified with an image densitometer Bio-Rad.
2
.
7
. In situ hybridization Tissue preparation was performed essentially as
described by Cox and Goldberg [33]. Nodules were fixed overnight in FAA [50 vv EtOH, 5
vv glacial acetic acid, 10 vv formaldehyde] under constant vacuum, dehydrated through a
graded ethanol series, and embedded in Paraplast Oxford, St. Louis. Mo. USA. Tissue sections 10
m m thick were applied to precleaned slide glasses
treated with Vectabond Vector Laboratories,
Burlingame, CA, USA. Pretreatment, hybridiza- tion and washing of slides were performed essen-
tially as described by McKhann and Hirsch [34], except for addition of RNase 30 mg RNase A
NTE buffer 1 ml in the course of washing. Anti- sense
and sense
RNA probes
for in
situ hybridization were prepared from linearized plas-
mids with
digoxigenin DIG-11-rUTP
Boehringer Mannheim according to manufactur- er’s instruction. Hybridization was performed at
42°C for 16 h. After posthybridization treatment and incubation with antidigoxigenin conjugated
with alkaline phosphatase, color development was allowed in a dark cabinet for approximately 1 – 2
days with substrate, and stopped by immersing the slides in TE pH 8.0. The sections were dehy-
drated through a graded ethanol series and then mounted with Permount Fisher Scientific, Fair
Lawn, NJ, USA.
3. Results and discussion
