Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlbl and Mtlb2) have been cloned and their nucleotide sequences determined.
Plant Molecular Biology 17: 335-349, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.
335
Synchronous expression of leghaemoglobin genes in Medicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate Philippe Gallusci, Annie Dedieu, Etienne P. Journet, Thierry Huguet and David G. Barker* Laboratoire de Biologic Molrculaire des Relations Plantes Microorganismes, INRA-CNRS, BP27, 31326 Castanet-Tolosan Crdex, France (*authorfor correspondence) Received 19 December 1990; accepted in revised form 23 April 1991
Key words:combined nitrogen, gene regulation, leghaemoglobin, Medicago truncatula, nitrogen fixation
Abstract
Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlbl and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18 ~o), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes [2]. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule ofM. truncatula. Whilst Mtlbl mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25 ~o of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlbl and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.
Introduction
As is the case for other eukaryotes, plants do not possess the means to reduce atmospheric nitrogen to a form which can be metabolized. Thus,
most plants rely on the presence of inorganic nitrogen in the soil, usually in the form of nitrate. However, as a result of the unique symbiosis which can occur between certain leguminous plants and gram-negative rhizobia (Rhizobium,
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X57732 (Mtlbl) and X57733 (Mtlb2).
336
Bradyrhizobium and Azorhizobium) such plants are provided with a source of inorganic nitrogen in the form of ammonia. This symbiosis occurs in specialized plant organs, known as root nodules, in which the rhizobium differentiates into the socalled bacteroid form, synthesizing the nitrogenase enzyme necessary for atmospheric nitrogen reduction (for review see [23]). When nitrates in the rhizosphere exceed a certain concentration, nodulation itself is inhibited, and nitrogen fixation is either reduced or totally switched off, suggesting that nitrates are the preferred nitrogen source [381. A number of plant proteins are synthesized uniquely in the root nodule, and these so-called nodulins [43] have been separated into two groups based on the timing of their synthesis. The early nodulins, synthesized during early stages of nodule formation, are thought to be involved either in the infection process (ENOD12 [35]) or in early nodule development (ENOD2 [ 10, 42]), whereas late nodulins, which are detected around the onset of nitrogen fixation, participate in the functioning of the nodule [44]. Leghaemoglobins belong to the group of late nodulins and are the major plant cytosolic protein within the nodule. These haemoproteins have a very high affinity for oxygen, and provide the necessary flux of this gas for the oxidative phosphorylation carried out by the nitrogen-fixing Rhizobium, whilst, at the same time, a low partial pressure of oxygen is maintained within the nodules which protects the oxygen-sensitive bacterial nitrogenase [1 ]. Leghaemoglobins are encoded by multi-gene families in those legumes which have been examined so far [2, 6, 21, 26]. In soybean there is very high sequence conservation between the four functional leghaemoglobin genes [7], and it has been shown that these genes are transcriptionally activated at different stages during nodule development [25]. In the case of the temperate legume alfalfa, we have been able to demonstrate that the leghaemoglobin gene family (a total of at least eleven bands on genomic Southern blots) can be subdivided into two groups based on differential hybridization with two alfalfa leghaemoglobin cDNA clones (pNL154 and pNL549) [2]. In
contrast with the situation for soybean, these two cDNAs differ by as much as 15~o at the nucleic acid sequence level, and furthermore, we have observed by northern analysis that transcripts hybridizing to a pNL154-specific probe and those hybridizing to a pNL549-specific probe are first detected at approximately the same stage of nodule development [2]. However, since the hybridization signals presumably corresponded to leghaemoglobin transcripts derived from more than one gene in each case, we could not conclude from this experiment that all leghaemoglobin genes are transcriptionally activated at exactly the same time. Because of the genetic complexity of alfalfa (a cross-fertile tetraploid) we chose to extend our studies on leghaemoglobin gene expression in Medicago using an autogamous diploid, Medicago truncatula, a plant selected as a model with which to study the symbiotic association with R. meliloti [3]. M. truncatula cv. Jemalong forms nitrogenfixing nodules with the laboratory strain of Rhizobium, Rm 2011, it has a genome which is half the size of that of alfalfa (0.9 pg/1C [8]) and, because of its self-fertile character, there is little, if any, genetic heterozygosity. We have previously shown that two analogous groups of leghaemoglobin genes exist in M. truncatula, and that the total number of hybridizing bands in genomic Southern blots is significantly less than for alfalfa [2]. In this article we describe the cloning and sequence analysis of a representative member from each of the two groups of M. truncatula leghaemoglobin genes, and by means of a highly specific RNAse protection assay we show that both genes are transcriptionally activated at precisely the same stage of nodule development. The inhibitory effect of combined nitrogen on symbiotic nitrogen fixation is of considerable agronomic importance, and has therefore been the subject of extensive research (for a recent review see [38]). Whereas very little is known about the molecular mechanisms involved, it has recently been shown that there is a significant reduction in levels of leghaemoglobin in the root nodules of a number of leguminous plants following the addition of combined nitrogen in the form of nitrate.
337 Since M. truncatula is particularly sensitive to the inhibition of nitrogen fixation by nitrate [ 12] we have examined to what extent leghaemoglobin gene expression is modified in response to exogenously supplied nitrate. We find that there is a significant reduction in levels of mRNAs corresponding to the two cloned leghaemoglobin genes concomitant with the drop in nitrogen fixation activity. However, we did not observe a reduction in the expression of the early nodule-specific gene ENOD2 following nitrate treatment. This difference presumably reflects the fact that the ENOD2 gene is expressed in the uninfected nodule parenchyma (inner cortex) [42], whereas leghaemoglobin transcripts are located in the infected cells of the nitrogen-fixing central tissue [5, 34].
Materials and methods
Plant material One-week-old seedlings of Medicago truncatula cv. Jemalong were grown in aeroponic conditions at 20 °C with a relative humidity of 7 5 ~ and a 14 h light/10 h dark photoperiod. Two days before inoculation with the Rhizobium meliloti strain RCR 2011 [30], the growth medium described by Lullien et al. [24] was replaced with fresh medium lacking the ammonium nitrate. Plants were generally grown for 2-3 weeks before inoculation in order to increase the yield of nodules per plant. When studying the effects of nitrate on nitrogenfixing nodules 5 mM potassium nitrate was added to the growth medium of plants which had been inoculated with R. meliloti 15 days earlier, and the nitrate-supplemented medium was renewed regularly to minimize variation in nitrate concentration. Nodules harvested at various times after inoculation with R. meliloti or after the addition of nitrate to nodulated plants were immediately frozen in liquid nitrogen and stored at - 70 oC. The nitrogen-fixing activity of nodules was measured by the acetylene reduction assay [41].
Construction of a M. truncatula genomic library and screening for leghaemoglobin clones A genomic library ofM. truncatula leaf DNA was constructed by ligating a Sau3A partial restriction digest (size-selected between 10 and 14 kb) into the Bam HI site of the phasmid vector pGY97 [45]. This library, which contains approximately 260000 recombinant clones with an average insert size of between 11 and 13 kb, and equivalent to three times the haploid genome of M. truncatula (9 × 105 kb), was divided into 26 sub-groups of 10000 clones. Plasmid DNA from each subgroup (2-3 #g) was digested with Eco RI, separated on 1~o agarose gels and transferred to GeneScreen membrane. Filters were then hybridized with specific probes derived from the two alfalfa leghaemoglobin cDNA clones pNL154 and pNL549 [2]. The sub-groups of the library containing inserts which hybridized with the leghaemoglobin probes were then plated out (approximately 1000 clones per plate) and screened by colony hybridization using Pall Biodyne nylon membranes according to the manufacturer's instructions. Hybridization with the alfalfa probes was carried out under reduced stringency conditions (37 °C, 50~o formamide)with subsequent washing in 2 × SSC at 60 °C.
DNA sequencing Restriction fragments covering the Mtlbl and Mtlb2 genes were subcloned in the vector pBScript SK + (Stratagene) using Escherichia coli TG2 [A(lac, pro), supE, thi, recA, srl::TnlO rcR, hsdA5(r- ,m - ), F' ( traD36, proAB +, lacI ~, lacZAM15] as the host strain. A series of overlapping unidirectional deletions were generated for both strands by controlled digestion with exonuclease III and subsequent S 1 nuclease treatment [14]. The deletions were subcloned into M13mpl0, M13mpll or M13mpl8 and dideoxy sequencing on single-stranded templates was performed essentially according to the method of Sanger etal. [33]. The junction sequences between two adjacent fragments were confirmed by
338 double-stranded sequencing of overlapping clones as described by Murphy and Kavanagh [281.
Northern analyses
Total RNA was extracted from roots, nodules and nitrate-treated nodulated roots as described in Lullien et aL [24]. For northern analysis, 5 #g of total RNA were separated in a 1 ~o agarose gel containing 6~o formaldehyde [31]. Transfer to GeneScreen membrane (NEN) and subsequent hybridization at 37 °C in the presence of 50% formamide and 10~ dextran sulphate were performed according to the manufacturer's instructions. Radioactive probes were prepared by the oligolabelling procedure [9] using (~-32p)-dCTP, and unincorporated nucleotides were subsequently removed by spin dialysis through Sepharose CL 6B (Pharmacia).
RNAse protection experiments
In order to provide the template for the Mtlbl antisense RNA probe a fragment extending from nucleotides - 109 to + 71 was isolated from an Exo III deletion construct and subcloned into pBScript SK +. For Mtlb2 a fragment extending from nucleotides - 53 to + 118 was subcloned into pGEM4 (Promega Biotec). Linear plasmids for in vitro run-off transcription were prepared according to Sambrook et al. [31], using 5' overhang restriction sites immediately following the insert. Radiolabelled RNA probes were synthesised using T7 RNA polymerase and including 50 #M (~-32p)-UTP (40 Ci/mmol) in the transcription buffer. Unincorporated nucleotides were removed by ethanol precipitation in the presence of 2.5 M ammonium acetate. For RNAse protection experiments 2 x 105 dpm of probe, either single or mixed, was hybridized with 2.5 #g of total RNA in 30/A of hybridization buffer (1 mM EDTA, 0.4 M NaCI, 40 mM Pipes-NaOH, pH 6.4, 80~o formamide). After heating to 85 °C for 10 min in order to de-
nature the RNA, the mix was incubated during 10 to 14 h at 37 oC. 0.3 ml of RNAse digestion buffer (10 mM Tris-HC1 pH 7.5, 300 mM NaC1, 5 mM EDTA, 40 #g/ml RNAse A) was then added to the hybridization mix and the whole incubated at 17 °C for 2 h. All other steps were performed according to Sambrook et al. [31 ].
Results
Isolation of two leghaemoglobin genomic clones of Medicago truncatula
By screening a genomic library of M. truncatula DNA (see Materials and methods) we were able to isolate clones hybridizing either to the alfalfa leghaemoglobin cDNA clone pNL549 or to the clone pNL154 (see Introduction). The first of these clones, with an insert size of approximately 12kb, encompasses the leghaemoglobin gene Mtlbl, the only M. truncatula gene which hybridizes to the pNL549-specific probe [2]. The second clone, with a genomic DNA insert of 13.6 kb, contains the gene Mtlb2, corresponding to one of the two major bands which hybridize with the pNL154-specific probe in genomic Southern analysis [2]. Figure 1 shows the location and orientation of the two leghaemoglobin coding regions within their respective genomic fragments. Both the size of the coding regions and the exon/ intron structure have been deduced from DNA sequence analysis (see below). DNA fragments covering the coding regions, the 3' non-coding regions and the immediate upstream promoter regions of both genes were subcloned into appropriate Bluescript vectors and unidirectional ExoIII deletions were generated on both strands for subsequent sequence analysis (see Materials and methods).
Comparison of the coding sequences of the two Medicago truncatula leghaemoglobin genes
Analysis of the nucleic acid sequences of the Mtlbl and Mtlb2 coding regions reveals that both genes
339
Mtlb 1 E P I ..... 1 .........................
B/Sou Sp H t ~ J
S I
II
B 1
I
I
XB I I
P I
E E J i
BHE I,.~jm~w
Mtlb2 EH B EB
j...i
II
H
EB
n
II
P
H H
n6 m i
i
H
n
S H
n
n
HPHB/Sau
n a n n..........................
P
E
i..., I
lkb
Fig. 1. Partial restriction maps for the genomic clones containing the M. truncatula leghaemoglobin genes, Mtlbl and Mtlb2. The positions and orientations of the two genes are shown relative to the adjacent stretches of cloning vector (thick black lines represent lambda EMBIA DNA and dotted lines represent pBR322 DNA). The locations of the open reading frames have been deduced from the sequence data, with filled boxes representing the exons and open boxes the introns. The direction of transcription is indicated for both genes by the arrow. B, Barn HI; B/San, Bam HI/Sau 3A junction; E, Eco RI; H, Hind III; P, Pst I; Sp, Sph I; S, Sst I; X, Xba I.
comprise four exons separated by three introns, encompassing 2.4kb and 1.3 kb respectively (Fig. 2). Mtlbl encodes a polypeptide of 146 amino acids and Mtlb2 a polypeptide of 145 residues, the difference of a single amino acid being due to an additional residue at position 21 in the Mtlbl protein. Significantly, we find exactly the same difference when we compare the sequences of the two alfalfa cDNA clones, pNL154 and pNL549 [2]. The homology between the coding regions of Mtlbl and Mtlb2 is 86~o at the nucleic acid level and only 82 ~o at the amino acid level, again similar to that between pNL154 and pNL549 [2]. Furthermore, as was to be expected from the hybridization data, there is a high level of conservation for the coding sequences between Mtlbl and pNL549 and between Mtlb2 and pNL154, equal to 95~o and 96~o respectively at the nucleic acid level. Perhaps even more strikingly, the 3' non-coding regions of the two alfalfa cDNAs are also highly homologous to the corresponding sequences of the M. truncatula genomic clones (95~o between Mtlbl and pNL549 and 88~o between Mtlb2 and pNL154). Taken together, these results suggest that the existence of these two groups of leghaemoglobin genes pre-
dates the common evolutionary ancestor of M. sativa and M. truncatula, and probably also of Melilotus, in which these two groups have also been detected [2]. The amino acid sequences of the putative leghaemoglobins encoded by Mtlbl and Mtlb2 were aligned with sequences of leghaemoglobins from various other plant species. Such a comparison reveals homologies, respectively, of 77 ~o and 76~o with LbI from pea [22], of 76~o and 75~o with the coding region of the Srglb2 gene from Sesbania rostrata [26], of 68~o and 69~o with Lbc3 from soybean [46] and of 39~o and 43 ~o with the haemoglobin from Parasponia [20].
The 5' and 3' non-coding regions of the Mtlb 1 and Mtlb2 genes
Analysis of the 5' non-coding regions of the Mtlbl and Mtlb2 genes reveals putative TATAAA boxes at position - 29 upstream from the transcription initiation site in both cases, and putative CAAT boxes at positions - 69 and - 63 respectively. In addition, the sequence motifs, AAAGAT and CTCTT, characteristic of 'late' nodulin genes
340
Mtlbl ACAAA•CTAGAAAATCAAATTTCATTGGAAAAATCT•ATAAAAAATGATAAAATATTGT••TTTTATAA•ATAAAAGACTAAATCGTTACAAAAAAAAGA298 -
TAAAGGAGC~AAAATGTTACCTAAAATTAAGTTAAG~GACTAATTATGTAATTTTGCCAAATTTTAAAGTTACTATCTTCGAGCTCGA~CCC~CTAT - 198 TGTAGCCGAAAAAGGGTATTATTTTATTTAATTGTAATTTATTTGCTTA~GTTTTTGAAAA~.FFF~-/~£GTCTCTTAATAACTACAATGGT~C~C~CA - 9 8 +1 AGCCATTATATTCTTTAAAAATAGAAT~TAATACACCTCTTTGCACCTCAAACTTTCT~wmvm1CAAAGGATGCATGTAACTTTATTGCA +3 TTCAAAATACAAATAATAAAACAAAAACAAGAAAAGAGAGAAATATGAGTTTCACTGATAAACAGGAGGCTTTAGTCAATAGCTCATATGAGGCATTC~ +103 S
F
T
D
K
Q
E
A
L
V
N
S
S
Y
E
A
F
K
T
ACAAAACCTTTCTGGTTATAGTGTTTTTTTCTACACTGTGTAAGTTTTATCTCTCGT~AGATTTTTTATTTGTTTTTGTCCTTTATGTTTATTGTC-F~q-~+203 Q
N
L
S
G
Y
S
V
F
F
Y
T
V
T
TAAGAAAGATACAATGTAAATGTAAATTAATTAGCAAAAATATGTTTTATTTTATTTTATTAAAATAGCATATTAGAGAAAGCACCTGC~~ +303 I
L
E
K
A
P
A
A
K
G
T
TGTTCTCTTTTCTTAAGGACTCAGCTGGAGTACAA~ATAGTCCTCAACTCCAAGCTCACGCTGAAA3~GTTTTTGGACTGGTAAGTATAATCTTACATCA +403 L
F
S
F
L
K
D
S
A
G
V
Q
D
S
P
Q
L
Q
A
H
A
E
K
V
F
G
L
ATTAATTTTTTTTCCTTTTATTCTTACAGTAATGTCAATGATTTTCTTGACATT~ATAATTATGAAATCTTTTTTTCTTTTGGTTACATAATTATGAAAA+503 CTTACTTTTATTTTCTTACTTTAAAATTTTAAGTTGATTAACATGATCCATATAATTATATTAAGAGAATTTGCAAGTTAGTTTAA~A~A~ ÷603 AAT~TTACTTCTTATATGCGTATGGCA~TACTTGTGTATCATTATTTATTTAATACGTATATATAATAGGTGTAAAAAAAAA~CCAT~.rL.n~ +703 TCCTCATGTTTTATTGTTGCAAATTAAATCTTCCAAGTATCTCAGTCGCTACATGCCT~CATCAAATTCTTAACAAACATTTTTAACTTCCAT~AT~C +803 AATCGCCACATCAAACTCTTTACAACCGTTTTTAACTCTAGG~GCCTACATCACCAATCAAAGTTGGCATCAACTATCCCATGA~C~ +903 CAATGCACATATGGAAGATTTAGAGTTTGAACCTCG~J%CACCGCA~AAAAAAAATCAACACCAACTTCAAAAG~TAATGTGGT~T~CT~CA +i003 TTCCTAAT~ATTTATATAGCAGATGCCCACAAATACTTTCATCAAGTTGCTAAGATATTTATTTGGTCTATGTTATACTTTTAATATGTAGCTTAAATCA ÷1103 ACATCAACTTAGAGTCATGTTTGCTCTTGTCTTAAAATATTAGATGATTAATGGATGGAGTGCCACAATATTAATTGTTGCAATTTGTGATATCTAGGAG +1203 ACCCAATTAACATAATTGAGATATAGAAGACAA/U~ATTCCACAATTGGAAGAAGAGACTTGATTAATATTCGTT~AATA.1Tr*-*~CTAGTTGGAAAATTG +1303 TTCAGTTCAAATTATAAATAGAACCAATACTTTATTTCTTGTGTTTTTTTTTAAGGATACTTCCGTTCTAGTAATAAGAATAATGTTA~ATTCGATTAG~ ,,1403
T
ATATTTTTTAACAACTTTCCTTGATTAACATAGTTATCAACCTAAAAATATTTTTAACTTTTAAATTATAGGTGCGTGATTCAGCTTCTCAACTTCGAGC +1503 V
R
D
S
A
S
Q
L
R
A
T
AACAGGGGGAGTAGTTTTGGG~GATGCTGCGTTGGGTGCTATCCACATTCAGAAGGGAGTTGTTGATCCTCATTTTGTGGTAT~T~TAT +1603 T
G
G
V
V
L
G
D
A
A
L
G
A
I
H
I
O
K
G
V
V
D
P
H
F
V
TTTTGTAGATGTATTTTAATTTTATTTTTTTGCATCCCCTAATTCRATAATACAAAGATAAATAAGCATATTTCATGCCACTTTTTATATTATGATATGG
+1703
AAGTA~TATTTCATGCCACTTTTTATTATCTGGAAGTGTGCTCCTCCTCGAGATCTCCGGTTCGATTCTCTCTGGTGTT~ATTTGGATGGGCTAATTTA
÷1803
GCTTCTTCAAATAATAATATAAACTCAATGGATTTACTAAAATTCTAAAAAGAATTGTGGATTTTTGTGATTTAGTTGTTTTTGTTCAGTATTATTTACT
+1903
CTGTTTTATAATTACTTTTATTTTAAATTATAAATAGTTACTGTCTATCAAATTTTATATCTRAAAAATGTTTCAGTCTGTTGTTTTTTCCTTCTTCTTC
+2003
CGGGAACTACATTTGTTACTTTTTCAAGACTAAAAGCCAATGATCCATATTGAAACAAATATCAATAATTTAATCTATATATTTGTTATGACAAAAAARA
+2103
TTATTTAAATTTACAATTGACTGTGTTCTGTCAATTAATAAGGAGGTAAAAATAGA~TAATACTAAAAATATATTAATAGTTCTTTTATTTGA~T TAATTAGTTTTCAATAAATACTTTCCATTTGTATTAACATACCTATTTTATAAGTGTTAAATCACTAATCCAAAGTTAATGTTTTTGGTGTCGTACAC'GT
÷2203
T
+2303 V
GGTTAAAGAGGCTTTGCTCRAGACAATAAAAG~AGCAGCAGGAGACAAATGGAGTGAAGAACTTAGCACTGCTTGGGAAGTAGCTTATGATGCACTGGCA V
K
E
A
L
L
K
T
I
K
E
A
A
G
D
K
W
S
E
E
L
S
T
A
W
E
V
A
Y
D
A
L
ACTGAAATTAAAAAAGCAATGAGTTAAACATGTGATGATCTATTATCATAAAAAGATTAATAAATAAAATATGTATTACTAAAACTTGTTAAACAAGTTC T
E
I
K
K
A
N
S
+2403 A +2503
*
CTA~TATGATAAA~ATATTTATGA~ATATTGTTACTTTGTTAGTGTTTATGTCGGTGAATCTCTTAGTCAAATCCCTaw1~TAw~CCCAAGTCA
+2603
AAGAGTACGTGTGTTCTATCACAATGTCTATTATATTAGTGAGGTACACATAAAATTCTAAGGCATTCTTCAATATCACAAAAAATCAAATTTq`TATTTC
+2703
TTCTGAAGTGGCCAATTGGTCCCATCATAGTAAAAAATTACAGTCATATTAGTAAACACCTTATAATGGTGTC&TTTAGTTTAGAAGTTTCGCCAATGTT
+2803
341
Mtlb2 A•TATATAACTCCA•AAAAATATAATTAATAAAATTATTTTAGTAAATGAAACTCATTTTGTTATTAAAAT•AAAATAACTCA•ATTATTAAAATTGGGC
- 312
ACAA~TcAAGTGAAATATTCACcATGAGAcGAGGAGAATAATTATTTTTATTAGATTAATTAATAATTACATTGATTAAATCAAGTTAAACAATAC~-L.C
- 212
ATTAATGATTAGTTTTTTATTTAAAATATTTATTAACTT•AATGAAAACTTGTTAGATTAAGTTTTTTAAATGATTATTGTcT•TTTAATAATGTCAACA
- Ii 2
G~ATTT~CA~AAGCCAATAGATTCTTTAAAAAAAAAAT~AT~GCATCTCTTTT~ACCTC~AAGTTTTCTA~AAGTATTGAAT
-12
+i GTA~AATTAATA~ATAGCAAATA~AAATAGCAAAAATAAAAACAAAAAGAAGAAGAAAAGAGATAT----~GTTTTACAGAGAAACAAGAGGcTTTAGTGAAT G
F
T
E
K
Q
E
A
L
V
+89
N
Y
AGcTCATGGGAATTATTTAAACAAAACCCTGGTAATAGTGTTTTGTTCTACA•TATGTAAGTTTGATATATTTTTTTGTGTTTGTGTTTT•ACTTT••GT S
S
W
E
L
F
K
Q
N
P
G
N
S
V
L
F
Y
T
+189
I
T TAATTGTCAGTTTGAAAGAGATTGTGTAAGTTAAAGAATAATTTGGTTATTTTGATTGAAATAGTATATTGGAGAAAC,CCCCTG•AGCAAAGC•GCATGTT I
L
E
K
A
P
A
A
K
G
M
T
+389
•TCTTTTCTTAAGGACAcAGCTGGAGTACAAGATAGTCCTAAA•TcCAAAGTCATGCTGAAAAAGTTTTTGC•AATGGTGAGTGTAATTAACATAAATTAA
S
F
L
K
D
T
A
G
V
Q
D
S
P
K
L
Q
S
H
A
E
K
V
F
G
+289 F
M
TAACTATGAATATAcTT•TTATTTGTTTTTTATTGTAATAGTTTATACTTATATTTTTTCTGTTAGGAcAAATCTATCATTATATATGCAATACCTT•AT
+489
TCCATTTAGTGTCATAACTATcCAAATTATAAAAAAAAATAAAAAAATCACATGTTTCAATAGTGCAAATTAGATCTTCCACAAATCTCGATTGTTATAA
+589
TAAAATTATGTATTACCTTCTCATTTTTAAGTT•A•GATCCTAcATATGA•CAATAGTGTAAATTTGATCTTCCACAAATCTCGATTGTTATAATCAGAA
+689
AATGGATTGATAATTTT~TTCTTAATAAAATATAATCACATTATAATAATAGCAATAATAAATGATTATAT~TLTrATTAACATGGACATTATCAATCAT
+789
T
AAGATATTTCTACTTTTTGAATTATAGGTC,CGCGATTCAGCTGTTcAACTCCGAC,CAACAGGGGGAGTAGTTTTGGGAGATC•CTACATTGGGTGCAATC• V
R
D
S
A
V
Q
L
R
A
T
G
G
V
V
L
G
D
A
T
L
G
A
T
AcATTCAGAAAGGAGTGGTTGATCCACATTTTGTGGTAAATATTTTAAAGGATGTGTTCTTAAGTTTTTTTTTAcTG•GTGGTTATTTATTCCATAATTC H
I
Q
K
G
V
V
D
P
H
F
+889
I +989
V
AATAAAATAACAATAAATAAGAAACTTCCATTTCATACcACATTTTTATATTATGATAAAAAAAAAAATAATAGTGGAATCATAAGCTATAACTAAAGAA
+1089
AATTATGTACAATTAAATTTGGAAGATAAAAcAATAGTAATATATAAAACATGATTTGATTAGTCGATATTGTAACATACATAGTTTCTAAGTGTTAAAT
+1189
T
CGTTAAT CCAAAGTTAATGTATTTGGTGTCTTGCAGGTGGTTAAAGAAGCTTTGCTGAAAACAATAAAGGAAGTATCAGGAGATAAATGGAGCGAAGAAT V
V
K
E
A
L
L
K
T
I
K
E
V
S
G
D
K
W
S
E
+1289 E
TGAGCACTGcTTGGGAAGTAGcCTATGATGcATTGGCAGCTGCAATTAAGAAGGcAATGGGTTAAATTT~TGATCTAGTTATAAAT~AATT~AATAA
L
S
T
A
W
E
V
A
Y
D
A
L
A
A
A
I
K
K
A
M
G
+1389
*
ATAAAAAATGTATTACTAAAACTTGTTTAAcAAGTTTCTAT~GTTGACTATTAAATATTGTTTGTGTTTATGCTACTATATCTCTTTATAAAAA
+1489
C T C C CTTLVAI~T~T*V%ACCTAATTCTAAAAGGGCTGAAACTAGTCAAAT A G T A T C T T C T T G T A T G G T T T C T G A A A G G A A A A A T A A T A G A T G A A A G A A A G T G A A
+1589
AAGAAAGAAAGTGAGAAGAAAGGAATCGCAAGCAGATCCCATCCAACATGTTTCTATCCAATTTGAACAAAAATAAATCTACCCAACATGTTTCATTCTT
+1689
TTTTATTTCGACGACGCAAAACAAGG
Fig. 2. Nucleotide sequences of the M. truncatula leghacmoglobin genes, Mtlbl and Mtlb2. In both cases nuclcotide positions are numbered relative to the transcription initiation sites (indicated by small open circles). For each gene putative TATAAA boxes, CAAT sequences and AATAAA polyadenylation signals are highlighted, and other sequences which are thought to play a role in polyadenylation are single-underlined (see text). The two sequence motifs common to the 3' non-coding regions of all leghaemoglobin genes are double-underlined. Vertical arrowheads indicate putative exon-intron boundaries, and the deduced amino acid sequences are presented directly underneath the corresponding exons. Initiation (ATG) and termination (TAA) codons have been single-underlined.
342 [32], and found in all the leghaemoglobin promoters so far examined [26, 36], are also present upstream of the Mtlbl and Mtlb2 coding regions (Fig. 3). As is the case for the Srglb2 gene [26], the AAAGAT motif is not 100~ conserved in either Mtlbl or Mtlb2. A 5' deletion analysis of the soybean lbc 3 promoter has provided evidence that a stretch of 40 bp, encompassing these two sequence elements, is in some way involved in nodule-specific transcription from this leghaemoglobin promoter in transgenic Lotus corniculatus [36, 37]. This entire cis element, called the organspecific element (OSE), is highly conserved in Mtlbl and Mtlb2 and similarly positioned with respect to the two TATAAA boxes (Fig. 3). Furthermore, a stretch of 16 nucleotides, present in Mtlbl (residues -165 to -180) and in Mtlb2 (residues - 177 to - 192) is about 80~o homologous to a sequence within the lbc3 promoter which has been shown to interact with protein
factors found in nodules of soybean [15] and in both nodules and leaves of Sesbania [26]. Two putative polyadenylation sites (AATAAA motifs) were found in the 3' non-coding regions of both genes. In the case of Mtlbl they are located 78 nucleotides and 158 nucleotides downstream from the TAA termination codon (Fig. 2) and are followed 39 and 20 nucleotides, respectively, further downstream by sequences which match the consensus YGTGTTYY, a motif which is often found approximately 30 nucleotides downstream from the AATAAA box of plant genes [16]. For Mtlb2, only the first of two AATAAA boxes (located 77 nucleotides and 142 nucleotides downstream from the TAA codon), is followed by a YGTGTTYY sequence (Fig. 2). The lengths of the 3' non-coding regions of the corresponding alfalfa cDNA clones [2] are consistent with recognition of the first AATAAA motif for both leghaemoglobin genes.
MtZbl
-243
TGC~AAATTTTAAAGTTACTATCTTCGAG~T~GAG~TCCC~T~TATTGTAGCCGAAAAAGGG~%TTAr1-rTATTTAAT~.
MtZb2
-245
T T A C A T T G A T T A A A T C A A G T T A ..... AACAATA. C T T T C A T T A A T G A T .... T A G T T T T T T ~ + T T A A A A T A T T T A T T
GmZbe3
-220
TTTTATATGGAAACTAAAAAAATATATATTAAAATTTTAAATTCAGAAT
MtZbl
-164
GT..
Mt Zb2
- 175
CTTCAATGAAAACTTGTTAGATTAAGTTTTTTW*v*~[m~ ~ATTGT~TAATA.
Gm Zbc5
-160
CTG~I~TGA
MtZbl
- 99
. CAAGCCATTATATTCTTTAAAAATAGAATTACAATAAAATAATA. CAC~TGCACCTCAAACTTT . CTATATAAAC
MtZb2
-101
. CAAGCCAATAGATTCTTTAAAAAAAAAATCATAAAAAATCAATTGCAT~TTCACCTCCAAGTTTTCTATATAAAC
GmZbc3
-85
ACAAGCCAAGAGAGAC
MtZbl
- 22
AAAGGATGCATGTAACTTTATTGCATTCAAAATACA
MtZb2
-22
AAGTATTGAATGTACAATTAATACATAGCAAATACA.AATAGCAAAAATA.AAAACAAAAA~AAGAAGAAAAGAGATATG
GmZbc3
-21
AAGTATTGGATGTGAAGTTGTTGCATA..ACTTGCATTGAACAATTAATAGAAATAACAGAAAAGTAGAAAAGAAATATG
AATTT
...... A T T T G C T T A A G T T T T T C , ~ z v A w ~ N m ~ T T T G T ~ .
.....
.... A A T ...... ~ T T T ~ T T A A A T T A T T T A
AATA. A C T A C A A
.....
TGGTCACCTCCA
A T G T C A A ..... C A G C C A T T T C C A
G'I'~2GATTT~GTT'I~I~Gt~II~ATTG'I~I~ACCATAC..C~TT~TCACCCTCCTCCA
....... ATAAGTTTTATTAGTTATTCTGATCA~
.... A A T A A T A A A A C A A A A A C
........ CAAGCCTTCTATATAAAT
. .A A G A A .A A G A G A G
.A A A T A T G
Fig. 3. Optimal sequence alignment of the Mtlhl, Mtlh2 and soybean Gmlhc 3 [36] promoter regions. Sequence identities bet~ccn M t l b l and Mtlb2 and between Mtlb2 and Gmlbc 3 are indicated with an asterisk. The A T G codons and the T A T A A A boxes are single-underlined. The Gmlbc 3 organ-specific element is double-underlined and the sequence motifs common to late nodulin genes are highlighted. The protein binding site 2 o f Gmlbc 3 [15] and the homologous regions of M t l b l and Mtlb2 have been boxed.
Nucleotides have been numbered with respect to the transcription start site.
343 In addition to these putative polyadenylation sites, AATAAA and AAAACTTGTT motifs, common to all leghaemoglobin genes so far examined, are also present in the 3' non-coding regions of Mtlbl and Mtlb2 (Fig. 2). Until now, the function of these highly conserved sequences remains unknown, although it has been proposed that they may play a role in stabilizing the leghaemoglobin mRNA [19].
The expression of Medicago truncatula leghaemoglobin genes during nodule development RNAse protection experiments were performed in order to confirm that both Mtlbl and Mtlb2 are transcribed in the root nodule, and to map their transcription initiation sites. The same technique was then used to compare, quantitatively, the expression of these two genes during nodule development. An antisense RNA probe corresponding to the
5' end of the Mtlbl gene was hybridized with total RNA purified from 9-day-old nodules. After RNAse treatment we observed a major protected fragment of 71 nucleotides (Fig. 4), which allowed us to map the principal transcription initiation site aproximately 29bp downstream of the TATAAA box. In addition to the major protected fragment there are also two minor protected RNA fragments of slightly greater length. Using the Mtlb2-specific probe, a major protected fragmentof 118 nucleotides is observed (Fig. 4), thus also placing the principal transcription initiation site approximately 29 bp downstream from the TATAAA box. As was the case for Mtlbl, two slightly larger protected fragments of low abundance are also detected, suggesting that a small percentage of both mRNA populations comprise transcripts which are 5-10 nucleotides longer than the major transcript. Precision in assigning the sites of transcription initiation is limited by the specificity of RNAse A, which only cuts after pyrimidine nucleotides. No signal was detected
Am Mtlb 1 T m
I-
k-
I
I
I
I I
16
I < .................
I I
180
.................
>
I
I 8
M tlb 2
I
I I I
l
I
< ................
I !
171
................
>
I
I 17
Fig. 4A. Transcription of Mtlbl and Mtlb2 in M. truncatula root nodules. Schematic representation of the two antisense RNA probes. Template D N A fragments of 180 nucleotides (Mtlbl) and of 171 nucleotides (Mtlb2) were cloned into pBluescript SK ÷ and into pGEM4 vectors respectively (see Materials and methods). Filled boxes represent the first exon, continuous lines the 5' non-coding regions and open boxes represent the vector polylinker regions. For both genes the position of the TATAAA box is shown, and the restriction sites used to linearize the vector prior to in vitro transcription are indicated. The sizes (in nucleotides) of the different elements of each probe are indicated below, and the direction of transcription by the T7 RNA polymerase is shown by the large arrows.
344
Fig. 4B. RNAse protection assay for leghaemoglobin mRNAs. 2.5 #g of the following RNAs were incubated with 2 x 105 dpm of the appropriate antisense probe(s), treated with RNAse A in order to digest single-stranded RNA, and then electrophoresed in a 6% polyacrylamide/7 M urea gel: yeast RNA with combined Mtlbl and Mtlb2 probes (lane a); total RNA from M. truncatula roots with combined probes (lane b); total RNA from M. truncatula 9-day-old nodules with Mtlb2 probe (lane c), Mtlbl probe (lane d) and combined probes (lane e). The sizes of the protected fragments are estimated from a DNA sequencing track shown in lane f.
with total RNA extracted from roots, confirming that M. truncatula leghaemoglobin gene expression is nodule-specific. In contrast to Mtlbl, Mtlb2 is only one member of a small group (2-3) of leghaemoglobin genes in M. truncatula which hybridize strongly with the alfalfa cDNA probe pNL154 [2]. Given the very high specificity of the RNAse protection technique, it is most likely that the major protected RNA fragment observed with the Mtlb2 anti-sense probe is derived solely from Mtlb2 mRNA. Having established that both leghaemoglobin genes were transcriptionally active, we now
wished to use the two probes together in the same experiment so that the kinetics of accumulation of both mRNAs during nodule development could be compared directly. Since the probes hybridize to equivalent regions of the Mtlbl and the Mtlb2 genes, it was necessary to test whether there is competition between them for the target mRNAs. The results shown in Fig. 4 indicate that there is no detectable difference between the signals obtained with either separate or mixed probes, showing that under the conditions of the experiment, there is little or no competition between the two probes. Total RNA was prepared from nodules harvested at various times after inoculation with Rhizobium meliloti and used in an RNAse protection assay with the two mixed leghaemoglobin antisense RNA probes. In both cases, leghaemoglobin transcripts can first be detected at day 5 (Fig. 5), with levels increasing very rapidly to reach a maximum either at day 6 (Mtlb2) or day 7 (Mtlbl). No significant variation is observed right up to day 28, which was the last time-point tested. In order to determine the relative amounts of the two mRNA species once maximum levels have been reached (7 days onwards), the specific activities of the protected RNA fragments were calculated and the amount of radioactivity present in
Fig. 5. Timecourse ofleghaemoglobin mRNAinduction during M. truncatula nodule development. 2.5 #g of yeast RNA (Y), total RNA from roots (R), and total RNA from nodules harvested between 4 and 28 days after inoculation with R. meliloti were hybridized with the combined Mtlbl, Mtlb2 probe and subjected to the RNAse protection assay described in the legend to Fig. 4.
345 each band was measured. The ratio of Mtlbl mRNA to Mtlb2 mRNA for each time point between day 7 and day 28 varies between 2.8 and 3.5, indicating that the mRNA of Mtlbl is approximately three-fold more abundant than that of Mtlb2. Effect of exogenous nitrate on the expression of Medicago truncatula leghaemoglobin genes
Nitrate has been shown to inhibit the earliest stages of nodulation (infection by Rhizobium, nodule primordium initiation) as well as nodule functioning and growth [38], but very little is known about the effect of nitrate on the expression of nodule-specific genes. With this goal in mind, we initially studied the effect of exogenous nitrate on the nitrogen-fixing activity of M. truncatula nodules, and then subsequently measured the levels of leghaemoglobin mRNA present in the nitrate-treated nodules. 5 mM potassium nitrate was added to the growth medium of plants that had been inoculated with R. meliloti fifteen days earlier, and the nitrogen-fixing activity of nodules, estimated by the specific acetylene reduction activity (ARA), was determined at various times after the addition of nitrate. As shown in Table 1, there is only a modest decrease in specific ARA after the first 24 h in the presence of nitrate, but after 2 days the nitrogen-fixing activity drops to around 25~o of its initial value. This reduced level remains approximately constant over the following 2-day period, suggesting that a new equilibrium has been established. Experiments carried out over longer periods (up to 9 days after nitrate addition) have
demonstrated that this is indeed the case (results not shown). We also noticed that, under these conditions, the cylindrical indeterminate nodule continued to elongate, suggesting that meristematic activity is not seriously impaired. For this reason, in addition to evaluating leghaemoglobin gene expression in these nitrate-treated nodules, we also examined if there were modifications in the levels of mRNA corresponding to the early nodule-specific gene ENOD2, a gene which is expressed specifically in the uninfected nodule parenchyma (inner cortex) [42]. Northern analysis was carried out on total RNA extracted from M. truncatula nodulated roots, harvested at various times during nitrate treatment. Hybridizations were carried out, consecutively, with a soybean ENOD2 probe, an alfalfa non-specific leghaemoglobin probe and a ubiquitin probe, this latter serving as an internal control for any variations in RNA loading or transfer efficiency. As shown in Fig. 6A, while the quantity of mRNA hybridizing with the ENOD2 probe remains at approximately the same level throughout nitrate treatment, the amount of leghaemoglobin mRNA decreases concomitantly with the nitrogen-fixing activity of the nodules. To examine if both groups of leghaemoglobin genes respond similarly to nitrate treatment, an RNAse protection assay was performed, as described above, using total RNA from nitrate-treated nodulated roots. In Fig. 6B, it can be seen that the levels of Mtlbl and Mtlb2 mRNA decrease at similar rates, indicating that the expression of these two genes is modified in a similar fashion by the addition of exogenous nitrate to nodulated nitrogen-fixing plants.
Table 1. Effect of exogenous nitrate on the acetylene reduction activity of M. truncatula root nodules. Time course showing the decrease in specific acetylene reduction activity in M. truncatula root nodules over the 4 day period following the addition of 5 mM KNO 3 to the growth medium. Values are expressed as a percentage of the initial activity, and represent the average of three independent experiments
Rate of acetylene reduction/nodule fresh weight
0h
7h
24 h
48 h
96 h
100%
100%
75 _+ 10%
25 _+10%
20 + 10%
346 Discussion
Fig. 6. Effect of exogenous nitrate on M. truncatula nodule-
specific gene expression. (a). 5/zg samples of total RNA extracted from nodulated roots of M. truncatula, harvested at various times (indicated in hours) following the addition of 5 mM KNO3 to the growth medium, were subjected to northern analysis as described in Materials and methods. The blot was successively hybridized with the following three probes: a soybean ENOD2 cDNA probe kindly provided by Ton Bisseling, Wageningen (1 kb PstI fragment from plasmid pENOD2 [ 10]); an alfalfa leghaemoglobin cDNA probe (0.5 kb Pst I fragment from plasmid pNL515 [2]); a human ubiquitin probe kindly provided by Ove Wiborg, Aarhus (0.65 kb Xho I fragment from plasmid pHUb 14-38 [47]). After each hybridization the blot was washed at 65 °C in 2 x SSC, 0.1% SDS prior to autoradiography, and then totally stripped before rehybridization. (b). 2.5/~g of the total RNA preparations described above (with the exception of the 72 h sample) were assayed for Mtlbl and Mtlb2 mRNAs using the mixed probe RNAse protection protocol described in the legend to Fig. 4.
The relatively small leghaemoglobin gene family of the diploid self-fertile M. truncatula can be divided into two groups based on the extent of hybridization with two alfalfa leghaemoglobin cDNA clones, pNL549 and pNL154, which we have previously demonstrated to diverge significantly [2]. We have cloned the unique gene hybridizing strongly with the pNL549-derived probe (named Mtlbl), and one of the genes hybridizing with the pNL154-derived probe (Mtlb2). RNAse protection experiments have been used to show that both Mtlbl and Mtlb2 are transcribed in nodules, but not in roots, and to localize the major transcription initiation site for both genes. Transcripts corresponding to both the Mtlbl and Mtlb2 genes can first be detected 5 days after inoculation, which, in the case of M. truncatula, corresponds to a stage of nodule development 1-2 days before the onset of nitrogen fixation. In this respect it is worth noting that M. truncatula nodulates and commences nitrogen fixation several days earlier than the alfalfa clone 11 × 8 used in our previous studies [2]. It is very likely that the other members of the Mtlbt2 group of genes, if functional, are also transcriptionally activated at this same stage of nodule development, as was suggested by our previous studies with alfalfa [2]. In M. truncatula nodules Mtlb2 mRNA reaches a steady-state level 6 days after inoculation, whereas Mtlbl mRNA only reaches its steadystate level at day 7. Differences in the rate of gene transcription and/or mRNA turn-over are presumably responsible for both this observation and the 3-fold higher steady-state levels of Mtlbl mRNA as compared with Mtlb2 mRNA. On the other hand, we do not observe any modification in the relative amounts of Mtlbl and Mtlb2 mRNA in maturing M. truncatula nodules. Thus, we have as yet been unable to uncover any significant differences in the timing of expression of the different leghaemoglobin genes present in Medicago throughout nodule development. This is intriguing since, despite the greater sequence homology between functional soybean leghaemoglobin genes [7], they appear to be subject to different
347 regulatory controls within the developing nodule as compared with Medicago genes. This might reflect the different structures and developmental patterns of the two types of nodules, soybean nodules being 'determinate' with transient meristematic activity, whereas alfalfa or M. truncatula nodules are 'indeterminate' with a persistent meristem. The inhibitory effect of exogenous nitrate on nitrogen fixation in legumes has been well documented (for review see [38]), but unfortunately little is known about the molecular mechanisms by which this occurs. In addition to inhibiting the nodulation process, nitrate is also able to inhibit the nitrogen-fixing activity of a fully functional nodule. In either case the degree of inhibition depends on both the inherent sensitivity of the plant and the nitrate concentration applied [ 12]. In our experiments with M. truncatula we have employed conditions under which the nitrogenfixing activity of the nodule is only partially inhibited, thus allowing us to examine the relationship between the effects of nitrate on nodule activity and on nodule-specific gene expression. M. truncatula is particularly sensitive to exogenously supplied nitrate [ 12], and we have shown that when nodules are exposed to 5 mM KNO 3 for 2-3 days the nitrogen-fixing activity decreases to a steady-state level representing only 20-25 ~o of its initial value. We have analysed the m R N A populations corresponding to the two leghaemoglobin genes Mtlbl and Mtlb2, and also to the gene ENOD2, which is thought to encode a cell wall hydroxyproline-rich glycoprotein involved in the growth and development of the basic nodule structure [10]. ENOD2 m R N A has been localized specifically in the nodule parenchyma (inner cortex) of soybean and pea nodules [42] and is also present in nodules that have not been infected with Rhizobium [11,40]. By contrast, leghaemoglobin transcripts are located in the infected cells of nodules [5, 34], and have not, so far, been detected in so-called empty nodules [ 11, 24, 40]. Thus one would predict that the regulation of these two nodule-specific genes would be quite different. Our findings clearly support such a conclusion, since ENOD2 gene expression is not
significantly altered bytreatingM, truncatula roots with 5 mM KNO3, whilst leghaemoglobin gene transcript levels drop dramatically, more or less in line with the fall in nitrogen-fixing activity. This effect is not exclusive to leghaemoglobin genes, since a similar response to nitrate treatment has been observed for transcripts corresponding to another late nodulin gene, NMS-25 of alfalfa [ 18] (results not shown). The m R N A levels of Mtlbl and Mtlb2 fall at approximately the same rate and to a similar extent, in response to nitrate treatment, indicating a common mechanism of inhibition for these two Medicago leghaemoglobin genes. However, we cannot rule out the possibility that the relative levels of the leghaemoglobin species themselves may be modified by nitrate treatment, as has been shown recently by Becana and Sprent [4] in the case of pea, soybean and mung bean nodules. Nitrate has also been shown to increase 5-fold the oxygen diffusion resistance of clover nodules [27 ]. Since the nodule parenchyma appears to be both the site for ENOD2 expression and the major site of diffusion resistance [48], van de Wiel et al. [42] have suggested that the ENOD2 protein may play an active role in the barrier to oxygen diffusion. If this were the case then one might expect that ENOD2 gene expression would increase significantly in response to nitrate treatment. Our failure to observe such an increase when M. truncatula nodules are exposed to 5 mM KNO3 (we estimate that variations in ENOD2 m R N A levels do not exceed 50~o in the experiment shown in Fig. 6) does not necessarily rule out a role for ENOD2 in the oxygen barrier. Further studies should perhaps focus on whether the quantity of the protein and/or its precise location within the nodule parenchyma is modified to combined nitrogen, or indeed other treatments which increase the oxygen diffusion barrier [48]. Various hypotheses have been put forward to explain how nitrate inhibits nitrogen fixation within the root nodule. Certain authors suggest that the nitrite produced in the first step of nitrate reduction could inhibit leghaemoglobin function [17, 29] and also nitrogenase activity [39]. Other theories propose that there is competition for
348 photosynthate-derived energy between the two energy-intensive pathways of nitrogen reduction [33] and, more recently, Heckmann etal. [13] have argued that the competition is for reducing equivalents. It is important to discover whether the changes in nodule-specific gene expression that we have observed are a consequence of one of these modifications in nodule metabolism, or whether the regulation of gene expression is itself a key event in the response to nitrate. We are now using an 'in situ' hybridization approach in order to determine in what way the distribution of leghaemoglobin transcripts is modified within the nitrate-treated nodule [5]. By such means we hope to be able to distinguish between effects on gene transcription and effects on leghaemoglobin mRNA stability.
Acknowledgements We would like to thank Eva Vincze and GyOrgy Kiss for their help in constructing the M. truncatula genomic library. We are also grateful to Charles Rosenberg for help in carrying out computer analyses of the sequences, to Danirle Rosenberg for typing the manuscript, and to Anne Stanford and other members of the laboratory for their constructive comments.
References 1. Appleby CA: Leghemogiobin and Rhizobium respiration. Ann Rev Plant Physiol 35:443-478 (1984). 2. Barker DG, Gallusci P, Lullien V, Khan H, Ghrrardi M, Huguet T: Identification of two groups of leghemoglobin genes in alfalfa (Medicago sativa) and a study of their expression during root nodule development. Plant Mol Biol 11:761-772 (1988). 3. Barker DG, Bianchi S, Blondon F, Dattre Y, Duc G, Flament P, Gallusci P, Grnier G, Guy P, Muel X, Tourneur J, Drnari6 J, Huguet T: M. truncatula, a model plant for studying the molecular genetics of the Rhizobiumlegume symbiosis. Plant Mol Biol Rep 8:40--49 (1990). 4. Becana M, Sprent JI: Effect of nitrate on components of nodule leghemoglobins. J Exp Bot 40:725-731 (1989). 5. de Billy F, Barker DG, Gallusci P, Truchet G: Leghemoglobin gene transcription is triggered in a single cell layer in the indeterminate nitrogen-fixingnodule of alfalfa. Plant J, in press (1991).
6. Bojsen K, Abildsten D, Jensen EO, Paludan K, Marcker KA: The chromosomal arrangement of six soybean leghemoglobin genes. EMBO J 2:1165-1168 (1983). 7. Brown GG, Lee JS, Brisson N, Verma DPS: The evolution of a plant globin gene family. J Mol Evol 21:19-32 (1984). 8. Essad S: Sur la relation possible d'une unit6 de variation saltatoire de I'ADN nucl6aire, le nuclron, et des chromosomes B dans le genre Medicago L. C R Acad Sci Paris srrie III 305:307-310 (1987). 9. Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 (1983). 10. Franssen HJ, Nap JP, Gloudemans T, Stiekema W, van Dam H, Govers F, Louwerse J, van Kammen A, Bisseling T: Characterisation of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development. Proc Natl Acad Sci USA 84: 44954499 (1987). 11. Govers F, Moerman M, Downie JA, Hooykaas P, Franssen HJ, Louwerse J, van Kammen A, Bisseling T: Rhizobium nod genes are involved in inducing an early nodulin gene. Nature 323:564-566 (1986). 12. Harper JE, Gibson AH: Differential nodulation tolerance to nitrate among legume species. Crop Sci 24:797-801 (1984). 13. Heckmann MO, Drevon JJ, Saglio P, Salsac L: Effect of oxygen and malate on NO3-inhibition of nitrogenase in soybean nodules. Plant Physiol 90:224-229 (1989). 14. HenikoffS: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351-359 (1984). 15. Jensen EO, Marcker KA, Schell J, de Bruijn FJ: Interaction of a nodule specific trans-acfing factor with distinct DNA elements in the soybean leghaemoglobin Ibc3 5' upstream region. EMBO J 7:1265-1271 (1988). 16. Joshi CP: Putative polyadenylation signals in nuclear genes of higher plants: A compilation and analysis. Nucl Acids Res 15:9627-9640 (1987). 17. Kanayama K, Watanabe I, Yamamoto Y: Inhibition of nitrogen fixation in soybean plants supplied with nitrate. I. Nitrite accumulation and formation of nitrosylleghemoglobin in nodules. Plant Cell Physiol 31:341-346 (1990). 18. Kiss GB, Vincze E, Vegh Z, Toth G, Soos J: Identification and cDNA cloning of a new nodule specific gene, Nms-25 (nodulin 25) of Medicago sativa. Plant Mol Biol 14:467-475 (1990). 19. Kuhse J, P~lhler A: Conserved sequence motifs in the untranslated 3' end of leghemoglobin transcripts isolated from broadhean nodules. Plant Sci 49:137-143 (1987). 20. Landsmann J, Dennis ES, Higgins TJV, Appleby CA, Kortt AA, Peacock WJ: Common evolutionary origin of legume and non-legume plant haemoglobins. Nature 324: 166-168 (1986). 21. Lee JS, Verma DPS: Structure and chromosomal ar-
349
22.
23. 24.
25.
26.
27.
28. 29.
30.
31.
32.
33.
34.
35.
rangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidization. EMBO J 3:2745-2752 (1984). Lehtovaara P, Lappalainen A, Ellfolk N: The amino acid sequence of pea (Pisum sativurn) leghemoglobin. Biochim Biophys Acta 623:98-106 (1980). Long SR: Rhizobium-legume nodulation: Life together in the underground. Cell 56:203-214 (1989). Lullien V, Barker DG, de Lajudie P, Huguet T: Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa). Plant Mol Biol 9:469-478 (1987). Marcker A, Land M, Jensen EO, Marcker KA: Transcription of the soybean leghemoglobin genes during nodule development. EMBO J 3:1691-1695 (1984). Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ: Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legume Sesbania rostrata: Conserved coding sequences, cis-elements and trans-acting factors. Mol Gen Genet 214: 181-191 (1988). Minchen FR, Minguez MI, Sheehy JE, Witty JF, Skot L: Relationship between nitrate and oxygen supply in symbiotic nitrogen fixation by white clover. J Exp Bot 37: 1103-1113 (1986). Murphy G, Kavanagh T: Speeding-up the sequencing of double-stranded DNA. Nucl Acids Res 16:5198 (1988). Rigaud J, Puppo A: Effect of nitrite upon leghemoglobin and interaction with nitrogen fixation. Biochim Biophys Acta 497:702-706 (1977). Rosenberg C, Boistard P, D~nari6 J, Casse-Delbart F: Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet 184:326-333 (1981). Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Sandal NN, Bojsen K, Marcker KA: A small family of nodule specific genes from soybean. Nucl Acids Res 15: 1507-1519 (1987). Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 (1977). Scheres B, van Engelen F, van der Knaap E, van de Wiel C, van Kammen A, Bisseling T: Sequential induction of nodulin gene expression in the developing pea nodule. Plant Cell 2:687-700 (1990). Scheres B, van de Wiel C, Zalensky A, Horvath B, Spaink H, van Eck H, Zwartkruis F, Wolters A-M, Gloudemans T, van Kammen A, Bisseling T: The ENOD12 gene product is involved in the infection process during the peaRhizobium interaction. Cell 60:281-294 (1990).
36. Stougaard J, Sandal NN, Gron A, Kflhle A, Marcker KA: 5' analysis of the soybean leghemoglobin lbc 3 gene: Regulatory elements required for promoter activity and organ specificity. EMBO J 6:3565-3569 (1987). 37. Stougaard J, Jorgensen J-E, Christensen T, Ktlhle A, Marcker KA: Inter-dependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters. Mol Gen Genet 220: 353-360 (1990). 38. Streeter J: Inhibition of legume nodule formation and N 2 fixation by nitrate. CRC Crit Rev Plant Sci 7:1-23 (1988). 39. Trinchant JC, Rigand J: Nitrite inhibition of nitrogenase from soybean bacteroids. Arch Microbiol 124:4%54 (1981). 40. Truchet G, Barker DG, Camut S, de Billy F, Vasse J, Huguet T: Alfalfa nodulation in the absence of Rhizobium. Mol Gen Genet 219:65-68 (1989). 41. Turner GL, Gibson AH: Measurement of nitrogen fixation by indirect means. In: Bergersen FJ (ed) Methods for Evaluating Biological Nitrogen Fixation, pp. 111-138. Wiley, Chichester (1980). 42. van de Wiel C, Scheres B, Franssen HJ, Van Lierop MJ, Van Lammeren A, Van Kammen A, Bisseling T: The early nodulin transcript ENOD2 is located in the nodule parenchyma (inner cortex) of pea and soybean root nodules. EMBO J 9:1-7 (1990). 43. van Kammen A: Suggested nomenclature for plant genes involved in nodulation and symbiosis. Plant Mol Biol Rep 2:43-45 (1984). 44. Verma DPS, Delauney AJ: Root nodule symbiosis: nodulins and nodulin genes. In: Verma DPS, Goldberg RB (Eds) Temporal and Spatial Regulation of Plant Genes, pp. 169-199. Springer-Verlag, Wien/New York (1988). 45. Vincze E, Kiss GB: A phosphate group at the cos ends of phage lambda is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, pGY97. Gene 96: 17-22 (1990). 46. Wiborg O, Hyldig-Nielsen J J, Jensen EO, Pahidan K, Marcker KA: The nucleotide sequences of two leghemoglobin genes from soybean. Nucl Acids Res 10: 34873494 (1982). 47. Wiborg O, Pederson MS, Wind A, Berglund LE, Marcker KA, Vuust J: The human ubiquitin multigene family: Some genes contain multiple directly repeated ubiquitin coding sequences. EMBO J 4:755-759 (1985). 48. Witty JF, Minchen FR, Skot L, Sheehy JE: Nitrogen fixation and oxygen in legume root nodules. Oxford Surv Plant Mol Cell Biol 3:275-314 (1986).
