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Keywords:

  • Pisum sativum;
  • alcohol dehydrogenase;
  • fructose-1;
  • 6-biphosphate aldolase;
  • in situ hybridization ;
  • nodule-enhanced malate dehydrogenase;
  • nodule-enhanced sucrose synthase;
  • phosphoenolpyruvate carboxylase;
  • root nodules
  • ADH;
  • alcohol dehydrogenase;
  • ALD;
  • fructose-1;
  • 6-biphosphate aldolase;
  • DAI;
  • days after inoculation;
  • Lb;
  • leghaemoglobin;
  • neMDH;
  • nodule-enhanced malate dehydrogenase;
  • neSS;
  • nodule-enhanced sucrose synthase;
  • PEPC;
  • phosphoenolpyruvate carboxylase

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Full length cDNAs encoding alcohol dehydrogenase (ADH), fructose-1,6-biphosphate aldolase (ALD), nodule-enhanced malate dehydrogenase (neMDH), phosphoenolpyruvate carboxylase (PEPC), and nodule-enhanced sucrose synthase (neSS) were isolated from a pea (Pisum sativum L.) root nodule cDNA library and characterized. Transcript abundance and cellular expression patterns for each gene were examined at different stages of nodule development. All the genes were expressed prior to the induction of nitrogenase suggesting a developmental signal as the initial trigger for expression. RNA tissue blots demonstrated that all the genes except ALD exhibit enhanced expression in effective nodules. In situ hybridization studies showed contrasting patterns of gene expression within various nodule zones. The highest expression of ADH was observed in interzone. ALD was expressed predominantly in nodule meristem, invasion zone and interzone. The neSS transcripts were found rather uniformly throughout the nodule. Expression of neMDH and PEPC was also detected throughout the nodule, but the highest levels were associated with interzone and N2-fixing zone.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Effective N2 fixation involves the complex interaction of legume plants with the soil bacteria, Rhizobium, Bradyrhizobium and Sinorhizobium. A new organ is formed from this interaction, the root nodule. Within the nodule, the bacteria reduce N2 to ammonia which the plant uses for amino acid and protein synthesis. In turn, the plant provides the bacteria with carbon for growth and N2 reduction. The carbon cost for this process is quite expensive and is estimated to require 5 to 10 g C per g N reduced ( Phillips 1980).

Although the ultimate source of C needed for N2 fixation is sucrose from photosynthesis, the nearly anaerobic (10 to 40 n M O2) nature of the nodule interior ( Tjepkema & Yocum 1974; Witty et al. 1986 ) results in sucrose being metabolized to organic acids, particularly malate, which are utilized by the bacteria to fuel nitrogenase ( Rosendahl, Vance & Pederson 1990; Salminen & Streeter 1992). Three critical enzymes involved in nodule organic acid biosynthesis are sucrose synthase (SS, EC 2·4.1·13), phosphoenolpyruvate carboxylase (PEPC, EC 4·1.1·31), and malate dehydrogenase (MDH, EC 1·1.1·37). Two other enzymes frequently induced during anaerobiosis, alcohol dehydrogenase (ADH, EC 1·1.1·1) and fructose-1,6-biphosphate aldolase (ALD, EC 4·1.2·13), may also be important in nodule C metabolism.

Nodule-enhanced forms of SS and PEPC have been documented and studied in several legume species ( Thummler & Verma 1987; Pathirana et al. 1992 , 1997; Küster et al. 1993 ; van Ghelue et al. 1996 ; Robinson et al. 1996 ; Zhang et al. 1997 ; Gordon & James 1997; Suganuma, Okada & Kanayama 1997; Hata, Izui & Kouchi 1998). However, less is known about the transcript temporal and cellular expression pattern for these genes. Recently a unique nodule-enhanced form of MDH has been reported for alfalfa (Medicago sativa L.) ( Miller et al. 1998 ), but its in situ expression pattern was not determined. Enzyme activity for ALD and ADH have been reported for soybean (Glycine max L. Merr.), pea (Pisum sativum L.) and chickpea (Cicer arietinum L.) ( De Vries, In’t Veld & Kijne 1980; Tajima & LaRue 1982; Suganuma & Yamamoto 1987; Kouchi et al. 1988 ; Anthon & Emerich 1990; Suganuma & LaRue 1993; Romanov et al. 1995 ; Thynn & Werner 1996), but genes encoding nodule forms of these enzymes have not been documented. To further define the factors affecting the expression of genes encoding the enzymes of pea root nodule C metabolism we thought it important to isolate and characterize the cDNAs encoding pea nodule ADH, ALD, MDH, PEPC, and SS. Gene expression in effective nodules was evaluated, both temporally and spatially by RNA blot analysis and in situ hybridization. These studies provide a foundation for subsequent analysis of gene expression in plant-controlled non-N2 fixing mutants of pea.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant material and bacterial strains

Pisum sativum seeds of line SGE ( Kosterin & Rozov 1993) were surface sterilized with sulphuric acid, imbibed overnight and germinated on Petri plates for 2 d. Seeds with emerged hypocotyls and roots were planted into sand and inoculated with effective Rhizobium leguminosarum CIAM-1026. The day of planting and inoculation was designated as day 0. Nodules were collected at 8, 12 and 33 d after inoculation (DAI) for the time-course experiments, and at 17 DAI for cDNA library preparation. For the tissue blot analysis, roots, stems, leaves and nodules were collected from plants at 19 DAI. All plant material was picked on ice and immediately used for RNA extraction. For in situ hybridization, nodules were harvested and immediately placed in fixation buffer.

Preparation of the pea root nodule cDNA library, isolation and characterization of the cDNA clones

A cDNA library was constructed in λZAPII vector (Stratagene, La Jolla, CA, USA) with poly (A)+ RNA from 17 DAI effective pea nodules according to the manufacturer’s directions. To isolate the corresponding pea cDNA clones, the library was screened with the following alfalfa DNA inserts: 700 bp polymerase chain reaction product of ADH, 1·4 kb ALD cDNA, 1·6 kb neMDH cDNA, 2·2 kb PEPC cDNA, and a 2·8-kb SS cDNA. Inserts were radiolabelled with 32P-dCTP with a random-primed labelling kit (Ambion, Austin, TX, USA). Plaque lifts were made onto BioTrace nitrocellulose membranes (Gelman Sciences, Ann Arbor, MI, USA). Hybridization at 50 °C and washing procedures were performed according to the manufacturer’s directions. All positively hybridized plaques were purified by two more rounds of screening. After isolation of pure plaques carrying the cDNAs of interest, the inserts were excised from the vector and their size determined by gel electrophoresis. The largest inserts for each type of cDNA were end-sequenced by the dideoxy termination method using Sequenase 2·0 (Amersham Pharmacie Biotec Inc., Piscataway, NJ, USA). Full length neMDH and neSS cDNAs were sequenced in their entirety by fluorescent automatic sequencing using ABIPrism 377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed using the GCG sequence analysis software (Genetics Computer Group, Madison, WI, USA).

RNA extraction and gel blot analysis

Total RNA was extracted from freshly collected tissues as described by Gregerson et al. (1993) . Poly(A)+ RNA for cDNA library construction was obtained by one cycle of oligo(dT)-cellulose chromatography.

For the gel blot analysis, total RNA (15 μg lane−1) was electophoretically separated on 1·5% agarose gels containing formaldehyde, transferred to ZetaProbe membranes (Bio-Rad Laboratories, Hercules, CA, USA) and hybridized to 32P-labelled probes as described previously ( Pathirana et al. 1992 ). The following pea cDNA inserts were used as the probes: ADH (1·4 kb), ALD (1·4 kb), MDH (1·5 kb), pepc/03 (1·1 kb), ss/1–3 (1·5 kb), and alfalfa leghaemoglobin MsLb3 (0·6 kb) and Sinorhizobium meliloti nifH (1·2 kb). Hybridizations were performed at 42 and 40 °C for homologous and heterologous probes, respectively. The data for RNA blots are representative of at least two separate experiments. Comparable RNA loading in each lane was confirmed by hybridization to alfalfa 28S-rRNA.

In situ hybridization

Freshly picked nodules were fixed overnight in 4% paraformaldehyde and 0·25% glutaraldehyde in 50 m M sodium phosphate buffer, pH 7·2. After rinsing several times with water, nodules were passed through a graded ethanol series (20, 30, 50, 70 and 100%), 50 and 100% xylene and embedded in Paraplast (Oxford Labware, St. Louis, MO, USA). The 10-μm-thick nodule sections were affixed to poly L-lysine-coated slides. Individual nodules were serial sectioned. One section was probed for leghaemoglobin (Lb) transcripts as a reference, and other adjacent sections were probed for the pea C metabolism enzyme transcripts of interest.

To generate RNA probes, the following cDNA inserts were used: original pea cDNA clones – 1·4 kb ADH and 1·4 kb ALD; pea subclones 1·5 kb ss/1–3, 1·1 kb pepc/03 and 0·7 kb mdh-E2, and a 0·6 kb alfalfa MsLb3.

To obtain sense and antisense 35S-labelled RNA probes, the cDNA inserts in pBluescriptSK+/– or pBluescriptKS+/– vectors were linearized with XhoI or NotI and transcribed in vitro with T3 or T7 RNA polymerases (Stratagene) according to the manufacturer’s directions.

In situ hybridization was performed in mineral oil at 42 °C, essentially as described by Fleming et al. (1993) . 3–5 × 105 cpm in 50 μL of hybridization solution were applied to each slide. Final wash was performed in 0·1 × SSC at 50 °C. Hybridization and washing conditions were similar for all probes, including the heterologous alfalfa Lb. Slides were exposed with the photoemulsion NTB2 (Eastman Kodak, Rochester, NY, USA) for 2 to 4 weeks, developed, and stained with 0·05% Toluidine Blue O. After dehydration, the sections were mounted in Permount (Fisher Scientific Co., Fair Lawn, NJ, USA) and viewed using a Labophot microscope optics (Nikon Inc., Garden City, NY, USA). A minimum of six nodules for each probe and each time point were evaluated. No hybridization signal was detected with any sense riboprobe used in the experiment.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Isolation and characterization of cDNA clones encoding nodule ADH, ALD, MDH, PEPC and SS

After screening the pea nodule cDNA library with heterologous clones from alfalfa, full-length clones encoding ADH, ALD, MDH, PEPC and SS were isolated and the inserts sequenced.

Sequencing of both 5′ and 3′ termini showed that pea nodule ADH, ALD and PEPC cDNAs were identical to those reported previously for Pisum (GenBank accession numbers X06281, X89829, D64037, respectively). The ADH cDNA sequence corresponds to the adh1 gene ( Llewellyn et al. 1987 ); the nodule ALD cDNA clone is identical to pea cytosolic aldolase clone aldcyt2 (GenBank accession number X89829), and the PEPC cDNA clone is identical to that encoding a nodule-enhanced PEPC ( Suganuma et al. 1997 ).

The full-length SS clone is 2749 bp long. Its 3′-end is identical to a partial cDNA clone (732 bp) for SS from pea seed coat (GenBank accession number X98598). The deduced amino acid sequence of our nodule SS (2418 bp, 806 amino acids) encodes a protein with the predicted molecular mass of 89·6 kDa. Comparative analysis of the deduced amino acid sequences of five different legume sucrose synthases ( Fig. 1) demonstrated that pea SS is highly homologous to nodule SS reported from broadbean (Vicia faba L.), alfalfa and alder (Alnus glutinosa L.) (99, 97 and 83% identities on amino acid level, respectively). It also has a high degree of homology with SS isolated from soybean nodules and mungbean (Vigna radiata Wilczek) seedlings (data not shown). By comparison, the deduced amino acid sequence of our pea nodule SS was only 71% identical to a previously reported sequence of pea ‘second sucrose synthase isoenzyme’ (GenBank accession number AJ030231). These data show that we have isolated a cDNA for another SS isoenzyme. Based upon its isolation from a nodule cDNA library and on the data from RNA blot analysis (next section) we designate it as a nodule-enhanced SS (neSS) cDNA.

image

Figure 1. . Comparison of the deduced amino acid sequences of the pea nodule-enhanced sucrose synthase (neSS) and sucrose synthases (SS) from different legume species. Identical residues are indicated by dots (·). Dashed lines (-) indicate gaps introduced in the sequences to maximize similarity. Amino acid position numbers are indicated to the right. Ps-neSS-Pisum sativum (pea) neSS reported in this paper. V.f.-Vicia faba (bean), M.s.-Medicago sativa (alfalfa), A.g.-Alnus glutinosa (alder)-sucrose synthases from the corresponding species (GenBank accession numbers X69773, AF049487, X92378, respectively). Ps-SSII – pea second SS isoenzyme (GenBank accession number AJ030231).

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Sequencing of pea MDH cDNA clone showed similarities with alfalfa neMDH, but did not match to any other known pea sequences, including chloroplast MDH. Therefore, MDH clone isolated from pea nodules (1510 bp long) represents a novel cDNA type for pea. It contains an open reading frame of 1194 nucleotides and encodes a 398 amino acid protein with the predicted molecular mass of 44 kDa. The deduced amino acid sequence of pea MDH is 93 and 91% identical, respectively, to that of alfalfa and soybean neMDH, and 74% identical to a recently reported ( Berkemeyer, Scheibe & Ocheretina 1998) Arabidopsis thaliana chloroplast NAD-MDH ( Fig. 2). However, comparison of the N terminal portions of pea MDH and alfalfa, soybean and Arabidopsis MDHs shows considerable divergence (only 76, 51 and 31% identities, respectively). The first 82 amino acid residues of alfalfa neMDH encode a putative pre-sequence, as evidenced by processing in Escherichia coli and similarity to processing sites in mitochondrial and glyoxysomal MDHs ( Miller et al. 1998 ). Assuming similar processing of the pea MDH protein, the pre-sequence is cleaved at residue 74. We have designated this pea nodule MDH as nodule-enhanced (neMDH) due to its high similarity to alfalfa and soybean neMDH, as well as its enhanced expression in root nodules (next section).

image

Figure 2. . Comparison of the deduced amino acid sequences of the nodule-enhanced malate dehydrogenases (neMDH). P.s.Pisum sativum neMDH reported in this paper, M.s.-Medicago sativa neMDH (GenBank accession number AF020273), G.m.-Glycine max neMDH (GenBank accession number AF068686), A.t.-Arabidopsis thaliana chloroplast NAD-MDH (GenBank accession number Y13987). Identical residues are indicated by dots (·). Dashed lines (-) indicate gaps introduced in the sequences to maximize similarity. Amino acid position numbers are indicated to the right. The arrow indicates the proposed cleavage site of pea neMDH.

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The complete sequences of pea neMDH and neSS are deposited to the GenBank under the accession numbers AF079850 and AF079851, respectively.

Analysis of ADH, ALD, neMDH, PEPC and neSS gene expression in various pea tissues

The isolated pea cDNA clones were used as probes to evaluate the expression of the corresponding genes in different pea tissues. Total RNA was extracted from roots, stems, leaves and nodules, electrophoretically separated, blotted onto membrane and hybridized to radiolabelled cDNA inserts ( Fig. 3). As expected, Lb gave a hybridization signal only with nodules reflecting its nodule specificity. Expression of neMDH, PEPC and neSS was higher in nodules than in other tissues and confirmed the nodule-enhanced nature of these genes. While the expression of ADH was also increased in nodules, its overall transcript abundance was considerably lower, compared with the other genes studied, and a clear signal on the X-ray film could be detected only after a longer exposure. In contrast to the other studied genes, expression of ALD was not enhanced in nodules, and the abundance of ALD transcripts did not vary much between the examined tissues.

image

Figure 3. . RNA gel blot analysis of leghaemoglobin (Lb), alcohol dehydrogenase (ADH), aldolase (ALD), nodule-enhanced malate dehydrogenase (neMDH), phosphoenolpyruvate carboxylase (PEPC) and nodule-enhanced sucrose synthase (neSS) gene expression in various pea tissues. Total RNA from root (R), stem (S), leaf (L) and nodule (N) tissue was separated by electrophoresis in 1·5% formaldehyde-agarose gel (15 μg lane−1), transferred to ZetaProbe membrane, and probed with each of the above cDNA inserts.

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ADH, ALD, neMDH, PEPC and neSS gene expression during the development of pea root nodules

Expression of ADH, ALD, neMDH, PEPC and neSS genes in developing nodules was evaluated by RNA gel blot analysis. By 8 DAI, the pea plants had formed small white nodules; however, nitrogenase activity, as measured by acetylene reduction, was not yet detectable. By 12 DAI, nitrogenase activity was already present (0·33 μmol C2H4 plant−1 h−1), and it had increased even more by 33 DAI (2·30 μmol C2H4 plant−1 h−1). Analysis of the expression of a bacteroid nifH gene which encodes the nitrogenase component II demonstrated that nifH transcripts were detected first in 12 DAI nodules, and thereafter remained constant (data not shown).

Total RNA was extracted from 8, 12 and 33 DAI nodules and hybridized to the pea probes, essentially the same as for the tissue blot experiment. As shown in Fig. 4, expression of Lb was just detectable in nodules 8 DAI; it increased significantly by 12 DAI, and remained high until 33 DAI. Expression of the other studied genes were readily detectable in 8 DAI nodules. The expression of neMDH and PEPC increased until 33 DAI, whereas the levels of neSS transcripts that were already high at 8 DAI, did not vary much further. In contrast to neMDH, PEPC and neSS, the expression of ADH was the highest in 8 DAI nodules, but then decreased, and by 33 DAI only a faint ADH message was detectable. Expression of ALD remained relatively constant during the entire period of nodule development.

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Figure 4. . RNA gel blot analysis of leghaemoglobin (Lb), alcohol dehydrogenase (ADH), aldolase (ALD), nodule-enhanced malate dehydrogenase (neMDH), phosphoenolpyruvate carboxylase (PEPC) and nodule-enhanced sucrose synthase (neSS) gene expression during nodule development. Fifteen micrograms of total RNA from nodules (8, 12 and 33 d after inoculation) was applied to each lane, separated by electrophoresis in 1·5% formaldehyde-agarose gel, transferred to ZetaProbe membrane, and probed with each of the above cDNA inserts.

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In situ localization of ADH, ALD, neMDH, PEPC and neSS expression in pea nodules

To localize the transcripts of ADH, ALD, neMDH, PEPC and neSS within the nodule, we performed in situ hybridization. Longitudinal sections of pea root nodules were hybridized to 35S-labelled antisense and sense RNA probes for each of these genes. To correlate the spatial distribution of the messages with a known nodulin, we also included a Lb probe in our study. Three time points of nodule development (8, 12 and 33 DAI) were analysed. Because there was no difference in the expression pattern observed between 12 and 33 DAI nodules, only the results of in situ hybridization from 8 and 33 DAI are shown in Fig. 5 (general view) and Fig. 6 (higher magnification of the interzone and the adjacent areas of 33 DAI nodules).

imageimageimage

Figure 5. . Localization of leghaemoglobin (Lb), alcohol dehydrogenase (ADH), aldolase (ALD), nodule-enhanced malate dehydrogenase (neMDH), phosphoenolpyruvate carboxylase (PEPC) and nodule-enhanced sucrose synthase (neSS) transcripts in 8 and 33 DAI (days after inoculation) nodules by in situ hybridization, general view. Nodules were sectioned longitudinally and hybridized with 35S-labelled antisense RNA probes for the above genes. Pictures of each nodule section were made in both dark and bright fields (labelled by two subsequent letters). In the dark field, transcripts are identified by bright dots (silver grains). Bright field pictures illustrate nodule zones: I-nodule meristem, II-invasion zone, *-interzone, III-N2-fixing zone, IV-senescent zone. Arrows in k, l, s, t, w, x show localization of the signal in nodule vascular bundles. Bars correspond to 500 μm. a, b, c, d – localization of Lb transcripts: a and b, 8 DAI nodules; c and d, 33 DAI nodules. e, f, g, h – localization of ADH transcripts: e and f, 8 DAI nodules; g and h, 33 DAI nodules. i, j, k, l – localization of ALD transcripts: i and j, 8 DAI nodules; k and l, 33 DAI nodules. m, n, o, p localization of neMDH transcripts: m and n, 8 DAI nodules, o and p, 33 DAI nodules. q, r, s, t – localization of PEPC transcripts: q and r, 8 DAI nodules; s and t, 33 DAI nodules. u, v, w, x localization of neSS transcripts: u and v, 8 DAI nodules, w and x, 33 DAI nodules.

image

Figure 6. .In situ localization of leghaemoglobin (Lb), alcohol dehydrogenase (ADH), aldolase (ALD), nodule-enhanced malate dehydrogenase (neMDH), phosphoenolpyruvate carboxylase (PEPC) and nodule-enhanced sucrose synthase (neSS) transcripts in the interzone and the adjacent areas. Dark and bright field pictures of nodule meristem, invasion zone, interzone, and beginning of N2-fixing zone of 33 DAI (days after inoculation) nodules were taken at higher magnification. Nodule zones identified are as in Fig. 5. Bars correspond to 100 μm. a, b, Lb; c, d, ADH; e, f, ALD; g, h, neMDH; i, j, PEPC; k, l, neSS.

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Description of zones observed in indeterminate alfalfa nodules ( Vasse et al. 1990 ), is applicable to those of pea. Typically, five zones are present in mature nodules ( Fig. 5): the nodule meristem (zone I), the invasion zone (zone II), the interzone (zone II-III, *), the N2-fixing zone (zone III), and the senescent zone (zone IV). Young pea nodules at 8 DAI are comprised of predominantly zones I, II and interzone II-III with few to no nitrogen-fixing cells. By contrast, nodules at 33 DAI have not only zones I, II, II-III, but a large zone III and occasionally a small senescent zone IV.

All the sense riboprobes, used as controls to confirm the specificity of hybridization, failed to show any labelling within the nodule tissues (data not shown).

Lb expression could be detected in 8 DAI nodules. These nodules already contain an interzone, where bacteria-bacteroid transformation takes place, and a very small zone III. In both 8 and 33 DAI nodules, the Lb transcripts were found in the interzone and in the infected cells of the N2-fixing zone ( Figs 5a, b, c & d; Figs 6a & b).

Although ADH was expressed at a low level throughout the entire N2-fixing zone ( Figs 5e, f, g & h), two distinct bands with enhanced ADH expression were also noted. One band was found in both young (8 DAI) and mature (12, 33 DAI) nodules, and corresponded to the interzone ( Figs 6c & d). The second band was not found in 8 DAI nodules, but was clearly detectable in nodules by 12 DAI (data not shown), and remained visible in nodules 33 DAI. This second band was located in the N2-fixing zone, where ADH transcripts were located in both infected and uninfected cells.

Although cytosolic ALD (aldcyt2) was expressed throughout the nodule, the highest expression was associated with the nodule meristem, the invasion zone, and the interzone ( Figs 5i, j, k & l). A strong signal was also detected in nodule vascular bundles. Within the N2-fixing zone, ALD transcripts were located in both infected and uninfected cells ( Figs 6e & f).

The greatest expression of neMDH was found in the N2-fixing zone. A hybridization signal was also detected in the nodule meristem, the invasion zone, the interzone, and the senescent zone ( Figs 5m, n, o & p; Figs 6g & h). neMDH transcripts were found in both the infected and uninfected cells of the N2-fixing zone.

The hybridization signal for PEPC was significantly increased in the interzone, and reached maximum in the N2-fixing zone. To a lesser degree, PEPC transcripts were also observed in the nodule meristem, the invasion zone, and the senescent zone ( Figs 5q, r, s & t; Fig. 6i, j). Both infected and uninfected cells of zones III and IV were found to contain PEPC transcripts. Some PEPC expression was also associated with the nodule vascular bundles.

In a similar manner to neMDH and PEPC, expression of neSS could be detected throughout the nodule ( Figs 5u, v, w & x). However, the maximal expression occurred immediately after the meristem in the invasion zone. The high levels of neSS expression remained relatively constant within the invasion zone, the interzone, and the N2-fixing zone ( Figs 6k & l). In a similar manner to ALD and PEPC, the neSS transcripts were also present in the nodule vascular bundles.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

In this report we document the isolation of two novel full-length cDNAs encoding pea nodule-enhanced enzymes that are involved in nodule C metabolism. The first encodes a nodule-enhanced MDH (neMDH). Although an enhanced MDH enzyme activity in pea nodules has been documented previously ( De Vries et al. 1980 ), and the existence of a nodule-enhanced form of MDH in pea was predicted by ion-exchange chromatography separation of MDH from roots and nodules ( Appels & Haaker 1988), no cDNA clone for pea neMDH has been yet characterized. Miller et al. (1998) recently reported the isolation and characterization of a neMDH cDNA for alfalfa nodules. Analysis of alfalfa neMDH kinetic characteristics and phylogenetic relationship with other types of alfalfa MDHs (cytosolic, plastid, glyoxysomal and mitochondrial) showed the uniqueness of neMDH, which seems to be responsible for strikingly high malate concentrations in N2-fixing nodules. Our sequence comparison data confirms the suggestion that neMDH forms a separate class of MDHs, and its interspecies similarity exceeds intraspecies ( Gietl 1992; Miller et al. 1998 ). Pea neMDH shares 93 and 91% identical amino acids with alfalfa and soybean neMDHs, respectively, and the identity between chloroplast and nodule-enhanced MDHs of pea is less than 28%. Interestingly, pea neMDH has a significant degree of homology with another chloroplast-targeted MDH, NAD-MDH from Arabidopsis thaliana ( Berkemeyer et al. 1998 ). Preliminary immunogold studies of alfalfa neMDH also localize the enzyme in nodule amyloplasts (Trepp, Litjens & Vance, unpublished). Therefore, pea neMDH appears to be another plastid-targeted form of the enzyme.

The second reported novel full-length cDNA encodes a neSS. Sucrose synthase has been studied in many plant species. In monocots SS is represented by two isoenzymes encoded by separate genes ( Guerin & Carbonero 1997). By comparison, only few dicots have been reported to possess more than one SS isoenzyme (or gene), for example, cucumber (Cucumis sativus L.) and potato (Solanum tuberosum L.) ( Gross & Pharr 1982; Fu & Park 1995). Only one form of SS (one type of cDNA) has been known for legume species. Using an alfalfa neSS cDNA as a probe, we isolated a full-length SS cDNA (2749 bp), which is identical to a previously isolated partial (732 bp) pea seed coat SS cDNA ( Déjardin et al. 1997 ). The deduced amino acid sequence of pea neSS is only 71% identical to a second pea SS isoenzyme (GenBank accession number AJ030231). Thus, pea appears to have at least two different forms of SS: one that is highly expressed in root nodules, and a second as yet not fully characterized form.

Sucrose synthase was among the first nodulins identified ( Morell & Copeland 1985; Thummler & Verma 1987), and thereafter has been characterized in several legume species ( Küster et al. 1993 ; Heim et al. 1993 ; Skøt et al. 1996 ; Zhang et al. 1997 ; Robinson & Vance, unpublished). The strikingly enhanced SS enzyme activity in nodules of both amide- and ureide-transporting legumes appears to reflect the universal mechanisms of sucrose cleavage in this organ. Moreover, loss of SS enzyme activity and transcript is closely correlated to loss of nitrogenase activity in environmentally stressed nodules ( Gordon et al. 1997 ).

Enhanced transcript expression in nodules observed for neMDH, PEPC, neSS and ADH is consistent with previously documented increases in the enzyme activities and protein content ( De Vries et al. 1980 ; Suganuma & Yamamoto 1987; Pathirana et al. 1992 ; Thynn & Werner 1996; Gordon & James 1997; Suganuma et al. 1997 ) and apparently reflects the importance of these enzyme for nodule functioning. However, although transcripts for all genes studied, excluding ALD, were enhanced in effective nodules, none appeared to require nitrogenase activity for initial induction. All were detected in 8 DAI nodules by both RNA blot and in situ hybridization detection techniques. At that time, neither nifH transcripts, nor nitrogenase enzyme activity can be detected in pea nodules. Expression of genes encoding enzymes of nodule C metabolism prior to nitrogenase activity implies that some factor associated with nodule development may trigger enhanced transcript synthesis. Our results further confirm previous studies showing Lb to be expressed prior to nitrogenase ( Bisseling et al. 1980 ), and are consistent with recent studies showing that Lb expression is activated very early in nodule development ( Heidstra et al. 1997 ). The heightened expression of PEPC and neMDH occurring after the onset of nitrogenase activity (12 and 33 DAI) is consistent with results obtained in alfalfa ( Pathirana et al. 1992 ; Miller et al. 1998 ), and suggest that additional signals associated with effective nodules, such as reduced levels O2 or high NH4+ concentration in the nodule interior may be important in plant gene expression. Moreover, the variations in expression patterns for transcripts shown in Fig. 3 indicate that not all genes involved in C metabolism are regulated in a similar manner.

Although in situ hybridization has been used to assess the spatial expression patterns of Lb, SS and PEPC in legume nodules ( Kardailsky et al. 1993 ; van Ghelue et al. 1996 ; Pathirana et al. 1997 ; de la Pena et al. 1997 ; Hata et al. 1998 ), cellular expression patterns for ADH, ALD and neMDH have not been evaluated. In this work, we analysed the in situ distribution of transcripts encoding ADH, ALD, neMDH, PEPC and neSS, as they correlate with expression of Lb used as a reference marker. Generally, the expression of neMDH and PEPC most closely approximated that of Lb showing highest expression in the interzone II-III and the N2-fixing zone III. Such expression patterns demonstrate the relationship of these particular enzymes of C metabolism and organic acid production to the process of N2-fixation in the effective nodule. In pea nodules, nitrogenase is expressed in the interzone II-III and in the zone III ( Brito et al. 1995 ). Transcripts for PEPC and neMDH were detected in both infected and uninfected cells of the N2-fixing zone. While a role for these enzymes in providing organic acids for infected cells is apparent, their role in uninfected and/or cortical cells is less obvious. However, Day & Copeland (1991) have proposed that sucrose is metabolized to malate in uninfected cells by SS, PEPC, and MDH and then transported into infected cells for use in nitrogen reduction and ammonia assimilation.

Analysis of the promoter for nodule PEPC in alfalfa along with immunogold localization of the protein suggest a role for this enzyme in uninfected cells to regulate the O2 diffusion barrier ( Robinson et al. 1996 ; Pathirana et al. 1997 ). The presence of neMDH in uninfected cells may be related to osmoregulation of cell size and/or additional production of malate for transport into infected cells, such as seen in the shuttling of malate in C4 species ( Sheehy, Minchin & Witty 1983; Hatch 1992).

Pea neSS showed high expression throughout the nodule at all stages (from 8 to 33 DAI). Nodules are strong sinks for photosynthate, and sucrose produced in legume leaves is rapidly metabolized to UDP-glucose and fructose in root nodules ( Reibach & Streeter 1983). Our in situ hybridization data indicate that sucrose cleavage is apparently occurring throughout the nodule whereas malate synthesis is predominantly located in the infected cell zone. Similar patterns of SS transcript distribution have been noted in other legume species ( van Ghelue et al. 1996 ; de la Pena et al. 1997 ). Likewise, immunogold localization studies showed SS protein in both infected and uninfected cells. However, gold particle density was two- to three-fold higher in uninfected cells ( Gordon, Thomas & Reynolds 1992; Zammit & Copeland 1993; Gordon, Thomas & James 1995).

The detection of PEPC and neSS transcripts in pea nodule vascular bundles extends the observations showing localization of these transcripts/proteins to vascular bundles of nodules in other legume species ( Gordon et al. 1992 ; 1995; van Ghelue et al. 1996 ; Robinson et al. 1996 ; Pathirana et al. 1997 ). This phenomenon may be related to the previously defined role of these enzymes in phloem loading and unloading, transport of assimilates, and tissue sink strength capacity ( Nolte & Koch 1993; Martin et al. 1993 ).

Pea nodule vascular bundles also contained significant amounts of ALD transcripts. High levels of ALD enzyme activity were observed in the bundle sheath strands surrounding vascular tissues in leaves of several plant species ( Campbell & Black 1982; Wurtele & Nicolau 1986), suggesting high capacity for glycolysis in these tissues.

In efforts to assess whether anaerobiosis may affect the expression of certain nodulin genes, we evaluated transcripts for two genes, ADH and ALD, known to be induced during anaerobiosis in maize (Zea mays L.) and rice (Oryza sativa L.) ( Umeda & Uchimiya 1994; Sachs, Subbaiah & Saab 1996). Sucrose synthase has also been reported to be up-regulated by anaerobiosis ( Ricard et al. 1991 ; Xue, Larsen & Jochimsen 1991). Oxygen electrode measurements have shown that O2 concentration in the interior of nodules is about 10 to 40 n M ( Tjepkema & Yokum 1974; Witty et al. 1986 ). Thus, genes susceptible to regulation by low O2 might be expected to be highly expressed in the nodule interior. Only ADH expression appears to be consistent with a pattern related to low O2 tension. The highest expression of ADH was detected in the nodule cortex, interzone II-III, with lower expression in the N2-fixing zone (zone III). By comparison, ALD and neSS expression were detected in the meristem and infection zone, as well as in zones of potential anaerobiosis, comparable to where ADH is expressed. Thus, the data from ALD and neSS probes used in this study do not support the hypothesis that these genes may be regulated by low oxygen. This inconsistency may be in part due to the fact that we used conserved regions of the cDNAs as probes, and thus may have detected more than one form of either ALD or SS. Unequivocal verification that anaerobiosis may or may not play a role in nodule gene activation will require in situ evaluation with 3′-untranslated region gene-specific probes for all transcripts in question.

The isolation of five pea cDNAs encoding nodule enzymes of C metabolism has given us the opportunity to map the distribution of transcripts within effective nodules. The fact that there were striking variations in transcript expression show that unique regulatory patterns exist for individual genes expressed in nodules. These expression patterns reflect varying functions for different cell types. For example, all nodule cells appear to have the potential to degrade sucrose; among them, there is a subset found in the nitrogen fixing area which is characterized by the enhanced production of malate, apparently for N2 reduction by bacteroids. Further analysis of cellular expression of transcripts for enzymes of C metabolism may allow us to determine whether anaerobiosis is a factor in gene regulation. In addition, the expression patterns of Lb, PEPC, neMDH, neSS, ALD, and ADH in effective pea nodules have laid the foundation for an assessment of the expression of these genes in ineffective associations.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The work was supported in part by NATO Grant No HTECH.LG 941423. This paper is a joint contribution from the Plant Science Research Unit, USDA, Agricultural Research Service, and the Minnesota Agricultural Experimental Station (Paper No. 99–1-1–13–0112, Scientific Journal Series). A nifH cDNA was generously provided by Dr M. Sadowsky. The authors are grateful to the University of Minnesota Imaging Center for technical assistance in the figure preparation. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of products or vendors that might also be suitable.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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  1. While this manuscript was in review, a mutation in the pea locus rug4 which directly affects the neSS gene expression was described (Craig et al. (1999) The Plant Journal17, 353–362). The largest reduction of SS activity in mutant plants (by 85%) was observed in root nodules where N2-fixation was significantly reduced or absent. This fact lands further support to the important role of neSS in the functioning of the effective nodules.