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The asparagine synthetase gene VfAS1 is strongly expressed in the nitrogen-fixing zone of broad bean (Vicia faba L.) root nodules
ELSEVIER
Plant
Science 124 (1997) 89-95
The asparagine synthetase gene VfASl is strongly expressed in the nitrogen-fixing zone of broad bean (Vicia faba L.) root nodules Helge Kiister a, Ulrike Albus a, Martin Friihling ‘, Svetlana A. Tchetkova Igor A. Tikhonovitch b, Alfred Piihler ‘-*, Andreas M. Perlick ” h All-Russia
’ Lehrstuhl jiir Genetik, Unicersitiit Bielrfeld, Post&h Research Institute fbr Agricultural Microbiology> Podbelsky
100 131, 33 501 Bielejtild, Grrma~~~~ Shmsee 3. I89 620 St. Prtershw,y Pd~kirr
Received 19 August 1996; received in revised form 9 December
1996; accepted
13 February
‘,
8, R~r.tsur
1997
Abstract
A full-length transcript sequence encoding the broad bean (b’icicr @a L.) asparagine synthetase (AS) VfASl was isolated from a nodule cDNA library. Sequence homologies indicated that VfASl belonged to the glutamine-dependent type of AS enzymes. The corresponding gene was highly expressed in root nodules and at a three-fold lower level in uninfected roots. Additionally, traces of VfASl transcripts were detected in epicotyl and stem tissues. Tissue-print hybridizations revealed that the VfASl gene was strongly expressed in the nitrogen-fixing zone III of root nodules. VfASl transcripts were absent from the meristem, the prefixing zone II as well as from peripheral nodule tissues. 0 1997 Elsevier Science Ireland Ltd. Kqwords:
Nodule
nitrogen
metabolism;
Nodule-specific
1. Introduction The symbiotic interaction of legume plants with rhizobia results in the formation of novel plant organs designated root nodules. Depending on the plant species, these nodules display either a determinate or an indeterminate type of development [3,7,15,27]. Cells of the central nodule tissue are * Corresponding 0167-9452,‘97/$17.00
author 0 1997 Elsevier
PII SOl68-9452(97)04607-4
cDNA
library:
Tissue-print
hybridization
infected by the endosymbionts [11,27], which subsequently differentiate into bacteroids capable of reducing atmospheric nitrogen. During this symbiotic nitrogen fixation, the ammonia released by the bacteroids is transported across the peribacteroid membrane by ammonia channels [24] and is incorporated into glutamine within the infected plant cells [25,27]. In temperate legumes forming indeterminate-type nodules, asparagine serves as the major transport form for reduced nitrogen
Science Ireland Ltd. All rights reserved.
90
H. Kiister et al. /Plant Science 124 (1997) 89-95
from the root nodules to other parts of the plant [19,21]. Asparagine is synthesised in an ATP-dependent reaction by the enzyme asparagine synthetase (AS, EC 6.354) which transfers an ammonia group from glutamine to aspartate resulting in the formation of glutamate and asparagine. All eukaryotic and most prokaryotic AS enzymes are of this glutamine-dependent type, whereas an ammonia-dependent AS is present in E. coli [16]. For Pisum satiuum, Tsai and Coruzzi [23] reported the isolation of two nodule-enhanced transcript sequences encoding the AS isoforms AS1 and AS2. Despite the importance of AS enzymes for the nodule nitrogen metabolism, the localization of AS transcripts within root nodules was not reported yet. We have previously isolated an incomplete cDNA encoding a broad bean (Vi& faba L.) AS by differential screening of a nodule cDNA library [17]. In this study, we present the sequence of a full-length AS transcript sequence and investigate the expression pattern of the corresponding gene in different broad bean tissues. Finally, the localization of AS transcripts in root nodules is reported.
2. Materials and methods
2.1. Plant material and cDNA
libraries
Viciu fuba L. cv. Kleine Thiiringer plants were grown in the field or in the greenhouse. Two days after sowing, the seedlings were inoculated with Rhizobium leguminosarum bv. viciae VF39 to obtain infected plants. Tissues used for Northern hybridization were harvested as follows: nodules (32 days after sowing), uninfected roots (32 days), leaves (32 days), seeds (90 days), epicotyls (8 days), stems (8 days) and flowers (60 days). Apart from the uninfected root tissue, which was harvested from greenhouse plants, all tissues were harvested from plants grown in the field. A nodule-specific cDNA library was constructed from poly(A) + mRNA isolated from V. fuba root nodules and was screened for full-length clones as previously described [ 131.
2.2. Isolation of nucleic acids and recombinant DNA
techniques
For cloning purposes and the construction of riboprobe vectors, the plasmids reported in [13] were employed. The isolation of recombinant phage and plasmid DNA was carried out using the ‘Lambda Kit’ (Qiagen) and the ‘Plasmid Kit’ (Qiagen) as recommended by the manufacturer. Overlapping sequencing clones were generated using the ‘double-stranded Nested Deletion Kit’ from Pharmacia according to the manufacturers instructions. The isolation of RNA from broad bean tissues was carried out as described previously [ 171.
2.3. DNA sequencing
and sequence
unulysis
Sequencing reactions were performed according to Zimmermann et al. [28] using the ‘AutoRead Sequencing Kit’ (Pharmacia). Sequencing gels were run on the ‘A.L.F. DNA Sequencer’ (Pharmacia) using sequencing gel mixes of standard composition. All sequences reported here were determined from both strands. Nucleic acid sequences were read using the ‘A.L.F. MANAGER V3.0’ software (Pharmacia) and analyzed using the PC/Gene software package (IntelliGenetics, release 6.8).
2.4. Northern
hybridizations
The amount of 30 pg of total RNA extracted from the tissues mentioned above was separated electrophoretically and blotted onto Hybond-N nylon membranes (Amersham) as described [I 71. stringent hybridizations and Probe labeling, washes were carried out according to [ 171.
2.5. Tissue-print
hybridizations
Longitudinal sections of mature broad bean root nodules (32 days after sowing) were printed on Hybond-N nylon membranes (Amersham) as described [13]. Prehybridization (2 h) and hybridization (16 h) was carried out at 68°C in the buffer reported by Ktister et al. [13] against digoxigenin-labeled antisense VfASl riboprobes. As a
H. Kiister et al. jl Plant Scicwce
124 (1997) 89-95
Fig. I. DNA and deduced amino acid sequence of a full-length VfASl transcript sequence. The deduced cDNA no. 5269 encoding the broad bean asparagine synthetase VfASI is printed above the DNA sequence. motif characteristic of glutamine-dependent asparagine synthetases is boxed. The sequence was deposited under the accession number 272354.
control, prints were hybridized against the corresponding sense riboprobes. Stringent washes and detections of hybridizing transcripts were carried out according to Ktister et al. [13]. Detections were stopped after 4 h and the filters were photographed using an Olympus SZ-PT stereo microscope. To relate hybridizing regions to nodule zones, sections used for tissue-printing were stained for starch as described [13]. Stained sections were rinsed in distilled water and photographed at the same magnification as the tissue-print filter.
91
amino acid sequence of The N-terminal ‘MCGI’ in the EMBL database
3. Results and discussion 3. I. Isolation of u jid-lmgtll broad bran AS VjASl
cDNA
cwcoding t/w
To study organ-specific gene expression in broad bean ( Vicia jkzba L. cv. Kleine Thtiringer) root nodules. we constructed a nodule-specific cDNA library from nodule mRNA using differential hybridization [17]. This library was divided into more than 40 groups by cross-hybridization experiments and sequence analysis [ 17.181. Two
92
H. Kiister et al. /Plant
cDNAs of the nodule-specific cDNA library displayed homologies to AS transcripts, but did not contain full-length coding regions [17]. To isolate full-length AS transcripts, a broad bean nodule cDNA library was screened [12] using a hybridization probe corresponding to position 194-662 of the sequence presented in Fig. 1. The hybridizing cDNA no. 5269 was cloned and sequenced (Fig. 1). This cDNA, as most other cDNAs from this nodule cDNA library, is missing a polyA tail, which is most likely due to an exonucleolytic digestion during cDNA synthesis or to an incomplete second strand synthesis. The open reading frame identified on cDNA no. 5269 specified a protein of 586 amino acids with a deduced molecular weight of 66.246, which, as the corresponding gene, was designated VfAS 1. Interestingly, cDNA no. 5269 contained a 5’ untranslated region of 117 bp, as compared to 40-80 bp usually present in eucaryotic transcripts [lo]. As reported for the human AS mRNA [4], the unusually long 5’ untranslated region of the VfASl transcript could participate in a translational regulation of VfASl gene expression.
Science
124 (1997) 89-95
(Fig. l), which was shown to be the glutamine binding site of AS enzymes [26], is conserved between the broad bean and all other eukaryotic glutamine-dependent AS enzymes. We infer that the VfASl protein is a glutamine-dependent AS of V. fizba corresponding to the AS1 isoform from pea. 3.3. VfASl transcripts are most abundant broad bean roots and root nodules
To investigate the occurrence of VfASl transcripts in different broad bean tissues, Northern blot hybridizations were carried out as described [12]. These experiments revealed that VfASl transcripts of 2.2 kb were abundant in broad bean nodules. A lower amount of VfASl transcripts was detected in uninfected roots, whereas only traces of VfASl transcripts were present in epicotyl and stem tissues (Fig. 3). Additional bands below 2.2 kb in the nodule and root lanes are
Vicia faba AS1 Pisum
3.2. The VfASl sequence is homologous glutamine-dependent ASS
sativum ASI
Lotus japonicus AS2
to
Lotus japonicus AS1 -I
Pisum sativum AS2 Asparagus
Searches of the current releases of the GenBank and EMBL databases revealed significant homologies to AS sequences from plant, animal, fungal and bacterial species (Fig. 2). Surprisingly, the VfASl sequence was found to be significantly more similar to the E. coli AsnB than to AS sequences from the fungus Saccharomyces or animals. As expected, no significant homologies existed between the VfASl sequence and the E. coli AsnA protein encoding an ammonia-dependent AS. Detailed sequence comparisons with the two AS isoforms of pea [23] revealed 97.3% identical amino acids with isoform AS1 and an identity of 85.6% with AS2. Therefore, the VfASl sequence corresponded to the isoform AS1 of pea. In addition, the broad bean VfASl sequence displayed 89.3 and 85.0% identical amino acids to two AS sequences from Lotus japonicus (sequence entries X89409 and X89410 from the EMBL data base). The ‘MCGI’ motif from the N-terminus of VfASl
in
Brasska
M&a/is
deracea
Arabidopsis
AS AS
thaliana AS
Zea mays AS Escharichia
cdi As.nB
Saccharomyces
carwisiaa
Rattus norwgicus Mesocricetus Criwtulus F
AS
AS
auf&us
AS
longicaudatus
AS
Homo sapiens AS Mus musculus AS
Fig. 2. Unrooted dendrogramm of an alignment of the VfASl sequence to 15 AS sequences from other organisms. AS sequences are from the legumes Vicia jbha (present paper), Lotus japonicus (EMBL accessions X89409 and X89410) and Pisum satioum [23]; the plant species Asparagus of$cinalis [6], Brassica oleracea (EMBL accession X84448), Arabidopsis thaliana [14] and Zea mays (EMBL accession X82849): the bacterium Escherichia coli [20]; the fungus Saccharomyces cereoisiae (EMBL accession X83099) and the animal species Rat&s noruegicus [9], Mesocricetus auratus [8], Cricetulus longicaudatus [2] and Mus musculus (GenBank accession U38940). In addition, the AS sequence from Homo sapiens [l] is included.
H. Kiistrr
et al. /Plant
Scirnce
c)3
124 (1997) 89- 95
3.4. The VfASl gene is strongly e.vprrssrd nitrogen-fixing zone III qf root nodules
2.2 kb-
Fig. 3. Expression of the VfASl gene in different broad bean tissues. Northern blot hybridization of 30 pg of total RNA from nodules, uninfected roots, leaves, seeds. epicotyls, stems and flowers of broad bean against a VfASl probe. The length of full-size transcripts identified is indicated.
most likely due to the presence of degraded VfASl transcripts, reminiscent of the situation observed for the broad bean sucrose synthase mRNA [12]. In RNA dot blot hybridizations according to Ktister et al. [ 121. the VfASl transcript level in uninfected roots was estimated to be three-fold lower as compared to nodules (data not shown). This shows that compared to the expression of the pea AS1 gene, which was induced 20-fold in nodules in relation to uninfected roots [23], the enhancement of VfASl expression in nodules was lower. No broad bean transcript sequence corresponding to the pea AS2 isoform was detected in our experiments, which might be due to an even lower level of induction of such an isogene in broad bean nodules. This could be the case, since the AS2 gene of pea is expressed only five-fold stronger in nodules as compared to uninfected roots in comparison to a 20-fold nodule-enhanced expression of the AS1 gene [23]. Alternatively, AS2 isoforms could be absent in broad bean.
in the
In order to detect the site of expression of VfAS 1 sequences within root nodules, tissue-print hybridizations were performed as described [13]. In these experiments, strong hybridization signals revealed the abundance of VfASl transcripts in the nitrogen-fixing zone III of root nodules (Fig. 4). Towards the interzone II-III, the intensity of hybridizing transcripts decreased. No hybridization signals significantly above the background level were observed in the nodule meristem and the prefixing zone II as well as in the peripheral the nodule cortex and parnodule tissues, enchyma. As expected from the expression of the
A
B
Fig. 4. Localization of VfASl transcripts m broad bean root nodules by tissue-printing. (A) Tissue-print hybridization according to [I31 of a longitudinal broad bean nodule section against a VfASl antisense riboprobe. (B) Microphotograph of the nodule section used for tissue-printing after staining for starch. Amyloplasts in cells of the nodule interzone II III strongly stained black. In addition. a lower amount of starch was present in cells throughout the nitrogen-fixing zone III. From a comparison of A and B. the predominant presence of VfASl transcripts in the nitrogen-fixing zone III with a decreasing tendency towards the interzone II III was concluded. Relevant nodule zones were labeled as follows: II. prefixing zone; III. nitrogen-fixing zone. An R denotes hybridization signals in the root section. The bar represents a distance of 800 rim.
94
H. Kiister et al. /Plant
VfASl gene in uninfected roots (Fig. 3) VfASl transcripts were also detected in the root tissue (Fig. 4). In control experiments using sense riboprobes, no significant hybridization signals were observed (data not shown). The predominant localization of VfASl transcripts in the nitrogen-fixing zone III of root nodules indicated a synthesis of the encoded AS VfASl during the nitrogen fixation phase of nodule development. This is in accordance with the regulation of expression of AS genes from Arabidopsis thaliana [ 141 and Zra nznys [5]. These genes were expressed in physiological conditions resulting in carbon deprivation and hence in high N:C ratios. This is also the case for nitrogenfixing cells of root nodules, where carbon skeletons are used for the primary fixation of ammonia in glutamine. Since alfalfa glutamine synthetase transcripts were also localized in the nitrogenfixing zone III [22], the localization of VfASl transcripts reported here indicates a tight spatial coupling of the primary nitrogen assimilation into glutamine and the synthesis of the transport intermediate, asparagine, in root nodules of temperate legumes.
Science
[5]
[6]
[7]
[8]
[9]
[IO]
[II]
[I21
Acknowledgements S.A. Tchetkova and I.A. Tikhonovitch acknowledge grants from the ‘Bundesministerium fur Ernahrung, Landwirtschaft und Forsten’ (BML).
[I31
[I41
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(Eds.). Proc. First Eur. Nitrogen Fixation Conf.. Officina Press. Szeged. 1994. pp. 225228. P.H.S. Reynolds. D.G. Blevins, M.J. Boland, K.R. Schubert and D.D. Randall, Enzymes of ammonia assimilation in legume nodules: A comparison between ureideand amide-transporting plants. Physiol. Plant., 55 (1982) 755260. M.A. Scofield. W.S. Lewis and S.M. Schuster, Nucleotide sequence of Eschericlziu coli asnB and deduced amino acid sequence of asparagine synthetase B. J. Biol. Chem., 265 (1990) 12895 12902. D.B. Scott, K.J. Farnden and J.G. Robertson, Ammonia assimilation in lupin nodules. Nature. 263 (1976) 703 705. S.J. Temple. J. Heard. G. Gamer, K. Dunn and C. Sengupta-Gopalan. Characterization of a nodule-enhanced glutamine synthetase from alfalfa: nucleotide sequence, in situ localization and transcript analysis. Mol. Plant-Microbe Interact.. 8 (1995) 218-227.
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[23] F.-Y. Tsai and G.M. specific expression
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Dark-induced
of two asparagine
and organ-
synthetase
Pimm satimrm. EMBO J., 9 (1990) 323-332. [24] SD. Tyerman. L.F. Whitehead and D.A. Day, nel-like
transporter
of N,-fixing
for NH4+ on the symbiotic
plants.
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[25] C.P. Vance and J.S. Gantt, Control of nitrogen carbon metabolism in root nodules. Phys. Plantarum. (1992) 266m 274. [26] G. van Heeke and SM. asparagine
synthetase
264 11989) 550335509. [27] D.P.S. Verma. Signals endocytosis
Schuster,
w/i.
in root nodule
of Rhi:obium.
[28] J. Zimmermann,
Expression
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Erfle, K. Stucky. T. Kristensen, W. Ansorge. A simplified protocol for fast plasmid DNA sequencing. Nucleic Acids Res..
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Plant
Science 124 (1997) 89-95
The asparagine synthetase gene VfASl is strongly expressed in the nitrogen-fixing zone of broad bean (Vicia faba L.) root nodules Helge Kiister a, Ulrike Albus a, Martin Friihling ‘, Svetlana A. Tchetkova Igor A. Tikhonovitch b, Alfred Piihler ‘-*, Andreas M. Perlick ” h All-Russia
’ Lehrstuhl jiir Genetik, Unicersitiit Bielrfeld, Post&h Research Institute fbr Agricultural Microbiology> Podbelsky
100 131, 33 501 Bielejtild, Grrma~~~~ Shmsee 3. I89 620 St. Prtershw,y Pd~kirr
Received 19 August 1996; received in revised form 9 December
1996; accepted
13 February
‘,
8, R~r.tsur
1997
Abstract
A full-length transcript sequence encoding the broad bean (b’icicr @a L.) asparagine synthetase (AS) VfASl was isolated from a nodule cDNA library. Sequence homologies indicated that VfASl belonged to the glutamine-dependent type of AS enzymes. The corresponding gene was highly expressed in root nodules and at a three-fold lower level in uninfected roots. Additionally, traces of VfASl transcripts were detected in epicotyl and stem tissues. Tissue-print hybridizations revealed that the VfASl gene was strongly expressed in the nitrogen-fixing zone III of root nodules. VfASl transcripts were absent from the meristem, the prefixing zone II as well as from peripheral nodule tissues. 0 1997 Elsevier Science Ireland Ltd. Kqwords:
Nodule
nitrogen
metabolism;
Nodule-specific
1. Introduction The symbiotic interaction of legume plants with rhizobia results in the formation of novel plant organs designated root nodules. Depending on the plant species, these nodules display either a determinate or an indeterminate type of development [3,7,15,27]. Cells of the central nodule tissue are * Corresponding 0167-9452,‘97/$17.00
author 0 1997 Elsevier
PII SOl68-9452(97)04607-4
cDNA
library:
Tissue-print
hybridization
infected by the endosymbionts [11,27], which subsequently differentiate into bacteroids capable of reducing atmospheric nitrogen. During this symbiotic nitrogen fixation, the ammonia released by the bacteroids is transported across the peribacteroid membrane by ammonia channels [24] and is incorporated into glutamine within the infected plant cells [25,27]. In temperate legumes forming indeterminate-type nodules, asparagine serves as the major transport form for reduced nitrogen
Science Ireland Ltd. All rights reserved.
90
H. Kiister et al. /Plant Science 124 (1997) 89-95
from the root nodules to other parts of the plant [19,21]. Asparagine is synthesised in an ATP-dependent reaction by the enzyme asparagine synthetase (AS, EC 6.354) which transfers an ammonia group from glutamine to aspartate resulting in the formation of glutamate and asparagine. All eukaryotic and most prokaryotic AS enzymes are of this glutamine-dependent type, whereas an ammonia-dependent AS is present in E. coli [16]. For Pisum satiuum, Tsai and Coruzzi [23] reported the isolation of two nodule-enhanced transcript sequences encoding the AS isoforms AS1 and AS2. Despite the importance of AS enzymes for the nodule nitrogen metabolism, the localization of AS transcripts within root nodules was not reported yet. We have previously isolated an incomplete cDNA encoding a broad bean (Vi& faba L.) AS by differential screening of a nodule cDNA library [17]. In this study, we present the sequence of a full-length AS transcript sequence and investigate the expression pattern of the corresponding gene in different broad bean tissues. Finally, the localization of AS transcripts in root nodules is reported.
2. Materials and methods
2.1. Plant material and cDNA
libraries
Viciu fuba L. cv. Kleine Thiiringer plants were grown in the field or in the greenhouse. Two days after sowing, the seedlings were inoculated with Rhizobium leguminosarum bv. viciae VF39 to obtain infected plants. Tissues used for Northern hybridization were harvested as follows: nodules (32 days after sowing), uninfected roots (32 days), leaves (32 days), seeds (90 days), epicotyls (8 days), stems (8 days) and flowers (60 days). Apart from the uninfected root tissue, which was harvested from greenhouse plants, all tissues were harvested from plants grown in the field. A nodule-specific cDNA library was constructed from poly(A) + mRNA isolated from V. fuba root nodules and was screened for full-length clones as previously described [ 131.
2.2. Isolation of nucleic acids and recombinant DNA
techniques
For cloning purposes and the construction of riboprobe vectors, the plasmids reported in [13] were employed. The isolation of recombinant phage and plasmid DNA was carried out using the ‘Lambda Kit’ (Qiagen) and the ‘Plasmid Kit’ (Qiagen) as recommended by the manufacturer. Overlapping sequencing clones were generated using the ‘double-stranded Nested Deletion Kit’ from Pharmacia according to the manufacturers instructions. The isolation of RNA from broad bean tissues was carried out as described previously [ 171.
2.3. DNA sequencing
and sequence
unulysis
Sequencing reactions were performed according to Zimmermann et al. [28] using the ‘AutoRead Sequencing Kit’ (Pharmacia). Sequencing gels were run on the ‘A.L.F. DNA Sequencer’ (Pharmacia) using sequencing gel mixes of standard composition. All sequences reported here were determined from both strands. Nucleic acid sequences were read using the ‘A.L.F. MANAGER V3.0’ software (Pharmacia) and analyzed using the PC/Gene software package (IntelliGenetics, release 6.8).
2.4. Northern
hybridizations
The amount of 30 pg of total RNA extracted from the tissues mentioned above was separated electrophoretically and blotted onto Hybond-N nylon membranes (Amersham) as described [I 71. stringent hybridizations and Probe labeling, washes were carried out according to [ 171.
2.5. Tissue-print
hybridizations
Longitudinal sections of mature broad bean root nodules (32 days after sowing) were printed on Hybond-N nylon membranes (Amersham) as described [13]. Prehybridization (2 h) and hybridization (16 h) was carried out at 68°C in the buffer reported by Ktister et al. [13] against digoxigenin-labeled antisense VfASl riboprobes. As a
H. Kiister et al. jl Plant Scicwce
124 (1997) 89-95
Fig. I. DNA and deduced amino acid sequence of a full-length VfASl transcript sequence. The deduced cDNA no. 5269 encoding the broad bean asparagine synthetase VfASI is printed above the DNA sequence. motif characteristic of glutamine-dependent asparagine synthetases is boxed. The sequence was deposited under the accession number 272354.
control, prints were hybridized against the corresponding sense riboprobes. Stringent washes and detections of hybridizing transcripts were carried out according to Ktister et al. [13]. Detections were stopped after 4 h and the filters were photographed using an Olympus SZ-PT stereo microscope. To relate hybridizing regions to nodule zones, sections used for tissue-printing were stained for starch as described [13]. Stained sections were rinsed in distilled water and photographed at the same magnification as the tissue-print filter.
91
amino acid sequence of The N-terminal ‘MCGI’ in the EMBL database
3. Results and discussion 3. I. Isolation of u jid-lmgtll broad bran AS VjASl
cDNA
cwcoding t/w
To study organ-specific gene expression in broad bean ( Vicia jkzba L. cv. Kleine Thtiringer) root nodules. we constructed a nodule-specific cDNA library from nodule mRNA using differential hybridization [17]. This library was divided into more than 40 groups by cross-hybridization experiments and sequence analysis [ 17.181. Two
92
H. Kiister et al. /Plant
cDNAs of the nodule-specific cDNA library displayed homologies to AS transcripts, but did not contain full-length coding regions [17]. To isolate full-length AS transcripts, a broad bean nodule cDNA library was screened [12] using a hybridization probe corresponding to position 194-662 of the sequence presented in Fig. 1. The hybridizing cDNA no. 5269 was cloned and sequenced (Fig. 1). This cDNA, as most other cDNAs from this nodule cDNA library, is missing a polyA tail, which is most likely due to an exonucleolytic digestion during cDNA synthesis or to an incomplete second strand synthesis. The open reading frame identified on cDNA no. 5269 specified a protein of 586 amino acids with a deduced molecular weight of 66.246, which, as the corresponding gene, was designated VfAS 1. Interestingly, cDNA no. 5269 contained a 5’ untranslated region of 117 bp, as compared to 40-80 bp usually present in eucaryotic transcripts [lo]. As reported for the human AS mRNA [4], the unusually long 5’ untranslated region of the VfASl transcript could participate in a translational regulation of VfASl gene expression.
Science
124 (1997) 89-95
(Fig. l), which was shown to be the glutamine binding site of AS enzymes [26], is conserved between the broad bean and all other eukaryotic glutamine-dependent AS enzymes. We infer that the VfASl protein is a glutamine-dependent AS of V. fizba corresponding to the AS1 isoform from pea. 3.3. VfASl transcripts are most abundant broad bean roots and root nodules
To investigate the occurrence of VfASl transcripts in different broad bean tissues, Northern blot hybridizations were carried out as described [12]. These experiments revealed that VfASl transcripts of 2.2 kb were abundant in broad bean nodules. A lower amount of VfASl transcripts was detected in uninfected roots, whereas only traces of VfASl transcripts were present in epicotyl and stem tissues (Fig. 3). Additional bands below 2.2 kb in the nodule and root lanes are
Vicia faba AS1 Pisum
3.2. The VfASl sequence is homologous glutamine-dependent ASS
sativum ASI
Lotus japonicus AS2
to
Lotus japonicus AS1 -I
Pisum sativum AS2 Asparagus
Searches of the current releases of the GenBank and EMBL databases revealed significant homologies to AS sequences from plant, animal, fungal and bacterial species (Fig. 2). Surprisingly, the VfASl sequence was found to be significantly more similar to the E. coli AsnB than to AS sequences from the fungus Saccharomyces or animals. As expected, no significant homologies existed between the VfASl sequence and the E. coli AsnA protein encoding an ammonia-dependent AS. Detailed sequence comparisons with the two AS isoforms of pea [23] revealed 97.3% identical amino acids with isoform AS1 and an identity of 85.6% with AS2. Therefore, the VfASl sequence corresponded to the isoform AS1 of pea. In addition, the broad bean VfASl sequence displayed 89.3 and 85.0% identical amino acids to two AS sequences from Lotus japonicus (sequence entries X89409 and X89410 from the EMBL data base). The ‘MCGI’ motif from the N-terminus of VfASl
in
Brasska
M&a/is
deracea
Arabidopsis
AS AS
thaliana AS
Zea mays AS Escharichia
cdi As.nB
Saccharomyces
carwisiaa
Rattus norwgicus Mesocricetus Criwtulus F
AS
AS
auf&us
AS
longicaudatus
AS
Homo sapiens AS Mus musculus AS
Fig. 2. Unrooted dendrogramm of an alignment of the VfASl sequence to 15 AS sequences from other organisms. AS sequences are from the legumes Vicia jbha (present paper), Lotus japonicus (EMBL accessions X89409 and X89410) and Pisum satioum [23]; the plant species Asparagus of$cinalis [6], Brassica oleracea (EMBL accession X84448), Arabidopsis thaliana [14] and Zea mays (EMBL accession X82849): the bacterium Escherichia coli [20]; the fungus Saccharomyces cereoisiae (EMBL accession X83099) and the animal species Rat&s noruegicus [9], Mesocricetus auratus [8], Cricetulus longicaudatus [2] and Mus musculus (GenBank accession U38940). In addition, the AS sequence from Homo sapiens [l] is included.
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et al. /Plant
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3.4. The VfASl gene is strongly e.vprrssrd nitrogen-fixing zone III qf root nodules
2.2 kb-
Fig. 3. Expression of the VfASl gene in different broad bean tissues. Northern blot hybridization of 30 pg of total RNA from nodules, uninfected roots, leaves, seeds. epicotyls, stems and flowers of broad bean against a VfASl probe. The length of full-size transcripts identified is indicated.
most likely due to the presence of degraded VfASl transcripts, reminiscent of the situation observed for the broad bean sucrose synthase mRNA [12]. In RNA dot blot hybridizations according to Ktister et al. [ 121. the VfASl transcript level in uninfected roots was estimated to be three-fold lower as compared to nodules (data not shown). This shows that compared to the expression of the pea AS1 gene, which was induced 20-fold in nodules in relation to uninfected roots [23], the enhancement of VfASl expression in nodules was lower. No broad bean transcript sequence corresponding to the pea AS2 isoform was detected in our experiments, which might be due to an even lower level of induction of such an isogene in broad bean nodules. This could be the case, since the AS2 gene of pea is expressed only five-fold stronger in nodules as compared to uninfected roots in comparison to a 20-fold nodule-enhanced expression of the AS1 gene [23]. Alternatively, AS2 isoforms could be absent in broad bean.
in the
In order to detect the site of expression of VfAS 1 sequences within root nodules, tissue-print hybridizations were performed as described [13]. In these experiments, strong hybridization signals revealed the abundance of VfASl transcripts in the nitrogen-fixing zone III of root nodules (Fig. 4). Towards the interzone II-III, the intensity of hybridizing transcripts decreased. No hybridization signals significantly above the background level were observed in the nodule meristem and the prefixing zone II as well as in the peripheral the nodule cortex and parnodule tissues, enchyma. As expected from the expression of the
A
B
Fig. 4. Localization of VfASl transcripts m broad bean root nodules by tissue-printing. (A) Tissue-print hybridization according to [I31 of a longitudinal broad bean nodule section against a VfASl antisense riboprobe. (B) Microphotograph of the nodule section used for tissue-printing after staining for starch. Amyloplasts in cells of the nodule interzone II III strongly stained black. In addition. a lower amount of starch was present in cells throughout the nitrogen-fixing zone III. From a comparison of A and B. the predominant presence of VfASl transcripts in the nitrogen-fixing zone III with a decreasing tendency towards the interzone II III was concluded. Relevant nodule zones were labeled as follows: II. prefixing zone; III. nitrogen-fixing zone. An R denotes hybridization signals in the root section. The bar represents a distance of 800 rim.
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VfASl gene in uninfected roots (Fig. 3) VfASl transcripts were also detected in the root tissue (Fig. 4). In control experiments using sense riboprobes, no significant hybridization signals were observed (data not shown). The predominant localization of VfASl transcripts in the nitrogen-fixing zone III of root nodules indicated a synthesis of the encoded AS VfASl during the nitrogen fixation phase of nodule development. This is in accordance with the regulation of expression of AS genes from Arabidopsis thaliana [ 141 and Zra nznys [5]. These genes were expressed in physiological conditions resulting in carbon deprivation and hence in high N:C ratios. This is also the case for nitrogenfixing cells of root nodules, where carbon skeletons are used for the primary fixation of ammonia in glutamine. Since alfalfa glutamine synthetase transcripts were also localized in the nitrogenfixing zone III [22], the localization of VfASl transcripts reported here indicates a tight spatial coupling of the primary nitrogen assimilation into glutamine and the synthesis of the transport intermediate, asparagine, in root nodules of temperate legumes.
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[5]
[6]
[7]
[8]
[9]
[IO]
[II]
[I21
Acknowledgements S.A. Tchetkova and I.A. Tikhonovitch acknowledge grants from the ‘Bundesministerium fur Ernahrung, Landwirtschaft und Forsten’ (BML).
[I31
[I41
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