A Soybean Plastid-Targeted NADH-Malate Dehydrogenase: Cloning and Expression Analyses

by John Imsande, Matthias Berkemeyer, Christine Gietl, Reid G. Palmer, Uwe Schumann
A Soybean Plastid-Targeted NADH-Malate Dehydrogenase: Cloning and Expression Analyses
John Imsande, Matthias Berkemeyer, Christine Gietl, Reid G. Palmer, Uwe Schumann
American Journal of Botany
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2Departments of Agronomy and of ZoologylGenetics, Iowa State University, Ames, Iowa 5001 1-1010 USA;
3Maltagen Forschung GmbH, Schaarstrasse 1, D-56626 Andernach, Germany;
4Pflanzenphysiologie, Fachbereich BiologielChemie, Universitat Osnabruck, D-49069, Germany;
5Technische Universitat Miinchen, Lehrstuhl fiir Botanik, Biologikum-Weihenstephan, Am Hochanger 4,
D-85350 Freising, Germany; and
6USDA-ARS-CICGR Unit and Departments of Agronomy and of ZoologyIGenetics Iowa State University,
Ames, Iowa 50011-1010 USA

A typical soybean (Glycine max) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2-ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyr- uvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule-enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana, which has a completely different transit peptide. In addition, the soybean nodule-enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of -34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene.

Key words: geranylgeranyl hydrogenase; Glycine max; malate dehydrogenase; metabolic regulation; nitrogen assimilation; nodule- enhanced malate dehydrogenase; pH stat.

Nodulated grain legumes possess two major sites of rapid genome encodes at least four PEP carboxylases, two of which nitrogen assimilation: (1) infected cells of the N,-fixing nod- are expressed in the seed (Hata, Izui, and Kouchi, 1998). Fur- ules and (2) pods and seeds during seed storage protein syn- thermore, in Viciafaba, enhanced nitrogen availability can in- thesis. Nitrogen assimilation by N,-fixing root nodules has duce the mRNAs for both a PEP carboxylase and legumin B, been studied extensively (Schuller, Turpin, and Plaxton, 1990; a major seed storage protein (Weber et al., 1998; Golombek Vance et al., 1994; Miller et al., 1998; Waters et al., 1998; et al., 1999). Also, an active MDH, whose function is similar Fedorova, Tikhonovich, and Vance, 1999; Trepp et al., 1999).

to the neMDH found in alfalfa (Miller et al., 1998) and pea A nodule-enhanced phosphoenolpyruvate (PEP) carboxylase nodules (Fedorova, Tikhonovich, and Vance, 1999), would be and a nodule-enhanced malate dehydrogenase (neMDH) in the required to facilitate OAA synthesis and metabolism during nodule provide oxaloacetate (OAA) to support synthesis of soybean seed filling. Malate dehydrogenase would catalyze the aspartate and asparagine and the continuation of the tricarbox- reduction of OAA to malate, thereby increasing net OAA syn- ylic acid (TCA) cycle. Because N can account for 6% of the thesis from PEP and providing malate to support the TCA dry mass of a soybean seed (Imsande, 1989), we reasoned that cycle, fatty acid biosynthesis, and other cellular activities grain legume seed formation may require enhanced activities (Plaxton, 1996; Golombek et al., 1999). These latter reactions of PEP carboxylase and MDH to support rapid synthesis of are important because the seed-enhanced PEP carboxylase is amino acids and seed storage proteins. Indeed, the soybean

strongly activated by glucose-6-phosphate, whereas aspartate, glutamate, and malate are inhibitors of the enzyme (Golombek I Manuscript received 13 March 2001; revision accepted 19 June 2001.

et al., 1999). In addition, oxalate is known to accumulate in

This work was supported by the Deutsche Forschungsgemeinschaft (Gi

developing soybean seeds (Ilarslan et al., 1997). Oxalate is a

15417-1). Joint contribution of the Iowa Agriculture and Home Economics

potent inhibitor of pyruvate kinase, an enzyme that competes

Experiment Station, Ames, Iowa (Journal Paper 5-18705; Projects 3352 and 3412) and the United States Department of Agriculture, Agricultural Research directly with PEP carboxylase for the utilization of PEP Service, Corn Insects, and Crop Genetics Research Unit. The mention of a (Smith, Knowles, and Plaxton, 2000). Coordination of these trademark or proprietary product does not constitute a guarantee or warranty

various metabolic activities is under stringent metabolic con-

of the product by Iowa State University or the USDA and does not imply its

trol and helps regulate the availability of precursor molecules

approval to the exclusion of other products that may also be suitable. Author for reprint requests (telephone: 515-294-7378; FAX: 515-294- needed for protein synthesis and seed maturation (Plaxton, 2299; e-mail: rpalmer@iastate.edu). 1996; Sakano, 1998; Weber et al., 1998; Golombek et al.,


1999). Sucrose synthase is reported to perform a vital role in this coordination process (Craig et al., 1999).

Many legumes produce a chloroplastic MDH that requires nicotinamide adenine dinucleotide phosphate (NADPH). Also, legumes produce nicotinamide adenine nucleotide (NADH)- dependent MDHs that are destined for the cytosol, glyoxy- somes, peroxisomes, mitochondria, and apparently the chlo- roplast (Gietl, 1992; Berkemeyer, Scheibe, and Ocheretina, 1998). Serologically, the mature glyoxosome and peroxisome MDHs are indistinguishable (Miller et al., 1998). Recently, a very active NADH-specific nodule-enhanced malate dehydro- genase (neMDH) was identified in alfalfa and pea nodules (Miller et al., 1998; Fedorova, Tikhonovich, and Vance, 1999). Low levels of neMDH were detected in most nonnodular tis- sue.

The broad objectives of our studies were to (1) clone and sequence several of the MDH genes present in soybean (Im- sande et al., 2001) and (2) identify, if possible, the function of each of the cloned MDHs through amino acid sequence analysis of the putative transit peptides and mature proteins, as well as the relative abundance of the mature protein in different tissues. The clone encoding the MDH described in this report was isolated from a soybean genomic library. This MDH lacks introns and is physically linked to geranylgeranyl hydrogenase (ggh), an enzyme involved in the synthesis of chlorophyll, tocopherol, and phylloquinone (Keller et al., 1998). The derived amino acid sequence of this soybean MDH is highly similar, in terms of transit peptide and mature sub- unit, to the neMDH from alfalfa, which also lacks introns (Miller et al., 1998). We show that the in vitro synthesized neMDH precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit. Be- cause the mature MDH subunit also is very similar to the plastidic NAD-dependent malate dehydrogenase from Arabidopsis (Berkemeyer, Scheibe, and Ocheretina, 1998), we used the respective Arabidopsis antibodies for western blot analysis of the different soybean tissues.


Isolation and characterization of genomic clones encoding a novel malate dehydrogenase and a geranylgeranyl hydrogenase-Genomic DNA of the soybean line T322 (Groose, Weigelt, and Palmer, 1988) was double-digested with EcoRI and XbaI and cloned into the EcoRI-site of the 1ambdaZAPII- vector (Stratagene, La Jolla, California). Preliminary experiments had shown that the mitochondrial MDH (mMDH) sequences did not contain an internal XbaI-site. The library was screened by hybridization to a watermelon mito- chondrial malate dehydrogenase cDNA clone (Gietl, Lehnerer, and Olsen, 1990). Positive phage clones were converted into the plasmid vector p-Bluescript SK by in vivo-excision according to the manufacturer's instructions and sequenced. In addition to several clones coding for mitochondria1 malate dehydrogenases (Imsande et al., 2001), one clone was identified with a 7.7- kb (kilobase) insert. It had an internal 4.8-kb XbaI-fragment coding for the C-terminal part of the geranylgeranyl hydrogenase and the full-length ne-MDH. Both proteins were read from the same DNA strand. This 4.8 kb XbaI fragment was flanked on both sides by a 2.5 kb EcoRI-XbaI fragment and by a 0.45 kb XbaI-EcoRI fragment, respectively. Due to our cloning strategy, we assumed that these EcoRI-XbaI fragments probably belonged to different parts of the genome and became linked to the internal XbaI fragment as cloning artifacts. To obtain the complete coding region of the geranylgeranyl hydrog- enase, the 530 bp (base pair) region of the 4.8-kb XbaI fragment that con- tained the partial coding sequence and the intron (bases 733 to 1262 of the final sequence GenBank accession number GBAN-AF068686) was used as a probe. (The prefix GBAN- has been added to each GenBank accession to link the online version of American Journal of Botany to GenBank but is not part of the actual accession number) After labeling with Digoxigenin-dUTP by polymerase chain reaction (PCR) amplification, the probe was used for screen- ing a commercial genomic library of 9 to 23 kb Sau3A partial restriction fragments of soybean DNA (Glycine max., cultivar Williams 79; Clontech, Palo Alto, California, USA) constructed in the lambda-FIXII-vector (Strata- gene). A 5.5-kb DNA fragment was sequenced and found to contain the com- plete coding region of geranylgeranyl hydrogenase and the complete gene of the neMDH.

In vitro transcription and translation of neMDH and import into isolated chloroplasts-The region encoding pre-neMDH corresponding to nt 3436- 4714 of the database entry GBAN-AF068686 was amplified via PCR using Pfu polymerase and the primer pair (5'-att gtt tgt atc aca ggc tga gat ggc agc- 3') and (5'-ccc gtt gaa aaa aaa tta agc agc aac agc-3'). (Start and stop codons are shown in boldface type.) The 1279-bp PCR-product was cloned into EcoRV digested pBSK, resulting in the clone pNEMDH8. After control se- quencing, pNEMDH8 was used for coupled in vitro transcription/translation using TNT Coupled reticulocyte Lysate System (Promega, Heidelberg, Ger- many) with T7-RNA-polymerase in the presence of (35S)methionine according to the manufacturer's instructions. 35S-labeled precursor protein was used for import experiments into isolated spinach chloroplasts as described (Weber et al., 1995). After pretreatment with thermolysin, the chloroplasts were recol- lected and fractionated into the envelope membranes and thylakoids and stro- ma according to Fliigge et al. (1989). The samples were subsequently ana- lyzed by SDS-PAGE (Laemmli, 1970) with acrylamide concentrations of 2.5% (stacking gel) and 12.5% (resolving gel) followed by autoradiography.

Protein extraction and western analysis-For preparation of clarified ex- tract, various soybean tissues were ground in liquid nitrogen and extracted with 1 mL of extraction buffer (20 mmollL Tris-HCL, 5 mM Na-ascorbate, 2 mmol/L EDTA, 1 mmol/L benzamidine, 1 mmol/L -amino-n-caproic acid,

0.5 mmol/L Pefabloc SC, pH 8.0) per gram of powdered tissue. The homog- enate was incubated on ice for 15 min and then centrifuged at 20000 X g for 20 min to remove cell debris. Protein was quantified (Bradford, 1976) using bovine serum albumin (BSA) as a standard, and aliquots of 50 pg protein were analyzed on 12% SDS-PAGE according to Laemmli (1970). Preparation of protein blots with subsequent immunodetection was as described elsewhere (Graeve, von Schaewen, and Scheibe, 1994). An isoform- specific polyclonal rabbit antiserum, raised against E. coli-expressed plastidic NAD-MDH from Arabidopsis (Berkemeyer, Scheibe, and Ocheretina, 1998), was used for the detection of NAD-MDH from soybean.


Cloning and sequence analysis of geranylgeranyl hydrogenase and a novel malate dehydrogenase-A soybean genomic library was screened using the watermelon mMDH cDNA (Gietl, Lehnerer, and Olsen, 1990) as a probe. A 4.8- kb fragment was detected, cloned, and sequenced. Sequence analysis revealed that this fragment encoded two proteins, the C-terminal portion of geranylgeranyl hydrogenase (ggh) and a complete MDH protein. Both proteins were read from the same DNA strand. Subsequently, the genomic library was suc- cessfully screened for the 5'-portion of the ggh gene and a continuous fragment composed of 5528 bp was isolated (AF068686).

In both the 4.8 and the 5.5 kb DNA fragments, the ggh coding sequence was interrupted by a single intron. This intron was composed of 309 bp and contained an imperfect 75 bp inverted repeat (i.e., 150 bp) that shows 63% identity. The AUG (translation start codon) for the ggh protein resided 93- 95 bp from the 5'-end of the coding strand of the 5.5-kb frag- ment. Hence, it is unlikely that the complete ggh gene has been isolated. The putative ggh protein encoded by the 5.5-kb

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160 170 180 190 200 210 220



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VGSLVRAYGLRREMEKIEA Mesembryanthemum cristallinum (GBAN-AF069318)

IGSLVRANALRREMDKLSV Nicotiana tabacum (GBAN-AJ007789)

IGSLVRANALRREIEKLSV Arabidopsis thaliana (GB AN-Y14044)
IGSLLRGNALAP-------Synechocystis sp. (GBAN-X97972)


Fig. 1. Alignment of the geranylgeranyl hydrogenases from Glycine max (GBAN-AF068686), Mesembryanthemunz cristallinum (GBAN-AF069318), Nicotiana tabacum (GBAN-AJ007789), Arabidopsis thaliana (GBAN-Y14044), and Synechocystis sp. (GBAN-X97972). Letters presented are the standard abbre- viations for the 20 amino acids, the order of which in the protein is indicated by the numbers. Identical amino acids are marked with an asterisk (*), similar amino acids (i.e., acceptable replacements) with a dot (.).

fragment was composed of 462 amino acids, -50 of which similar to the ggh produced by Synechocystis (GBAN

constitute an apparent transit peptide (GBAN-AAD28640). CAA66615; Addlesee et al., 1996) (Fig. 1). Although the pu-

The mature putative protein encoded by the 5.5 kb fragment tative transit peptides for the geranylgeranyl hydrogenases

(i.e., amino acid residues 51-462) was 91% identical and 95% from Arabidopsis, tobacco, and soybean were all 44-50 amino

similar to the putative ggh protein from tobacco (GBAN- acids residues in length and rich in serine, their sequence sim-

CAA07683), 87% identical and 94% similar to the ggh from ilarities were generally 40%.

Arabidopsis (GBAN-CAA74372; Keller et a]., 1998), 87%

identical and 92% similar to the ggh from Mesembryanthemum Properties of the plastidic MDH-The AUG start codon

ctystallinum (GBAN-AAC19396), and 68% identical and 79% for the novel plastidic MDH was located 1678 bp downstream


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........................... ************* * ****************

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************* *** ........................... **************

310 3 2 0 330 3 4 0 350 360


....................................... ....................

370 380 390 4 0 0 410 420


................................... ******.******.*.*

Fig. 2. Alignment of the nodule-enhanced malate dehydrogenases (neMDH) from Glyine max (GBAN-AF068686), Medicago sativa (GBAN-AF020273), and Pisum sativum (GBAN-AF079850) with plastid NAD-dependent malate dehydrogenase from Al-abidopsis (Y13987). Letters presented are the accepted abbreviations for the 20 amino acids, the order of which in the protein is indicated by the numbers. For the transit peptides of the three neMDHs, identical amino acids are indicated by an asterisk (*) above the sequence, whereas similar amino acids (i.e.. acceptable replacements) are indicated by a dot (.).Symbols for identical and similar amino acids for all four transit peptides and the four MDHs are shown below the sequences.

from the UGA (translation stop codon) for ggh. An imperfect to the putative neMDH from Pisum sativum (GBAN226-bp inverted repeat (i.e., 552 bp), which showed 59% iden- AAC28106), 91% identical and 96% similar to the neMDH tity, resided approximately midway between the ggh stop co- from Medicago sativa (GBAN-AAB99757), 87% identical and don and the malate dehydrogenase AUG start codon. The 94% similar to the chloroplastic NAD-MDH from Arabidopsis AUG start of MDH was located at 3468 bp. The UAA stop (GBAN-CAA74320; Berkemeyer, Scheibe, and Ocheretina, codon is located at 4707 bp. As indicated by alignment with 1998), and 65% identical and 78% similar to the glyoxysomal other MDH sequences, no introns were present in this 1239 MDH from soybean (GBAN-P37228; Guex et al., 1995). Only bp reading frame. Thus, a putative protein of 413 amino acids two transit peptides found in GenBank showed similarity to was encoded by this MDH gene. Alignment with other MDH that of the novel MDH described in this report. The 95-amino sequences indicated that the putative MDH protein bears a acid transit peptide of the novel MDH was 64% identical and transit peptide of -95 amino acids, whereas the mature protein 72% similar to that of the neMDH from Medicago sativa and would contain 3 17 amino acids residues (GBAN-AAC24855). 53% identical and 58% similar to that of the neMDH from The mature MDH protein was 92% identical and 96% similar Pisum sativum (GBAN-AAC28106) (Fig. 2). On the other

from the UGA stop codon of ggh. Although the deduced ami- no acid sequence of this MDH is 92% identical to that of the putative neMDH from pea (GBAN-ACC28106; Fedorova, Tikhonovich, and Vance, 1999) and 91% identical to that of alfalfa (GBAN-AAB997.57; Miller et al., 1998), its functions remain speculative. Nodulated pea and alfalfa plants are amide (i.e., asparagine) transporters, whereas nodulated soybean plants are ureide transporters. The reaction sequence for am- monia assimilation in nodulated pea and alfalfa would require a rapid rate of asparagine synthesis (Fedorova, Tikhonovich, and Vance, 1999; Trepp et al., 1999), whereas a nodulated soybean plant would require a rapid rate of ureide synthesis that occurs predominantly in the plastids (Atkins and Beevers, 1990). Ureide synthesis requires glutamine for the synthesis of phosphoribosylamine, glycine for the synthesis of glyci- namide ribonucleotide, a second molecule of glutamine for the synthesis of formylglycinamidine ribonucleotide, aspartate for the synthesis of aminoimidazole ribonucleotide, and two mol- ecules of serine as formyl donors (Atkins and Beevers, 1990).

In most plant tissues, primary ammonium assimilation is accomplished by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT). Nodule-enhanced iso- forms of both enzymes occur in several legumes. In contrast to the ferredoxin-dependent (Fd) GOGAT of photosynthetic tissues, the nodule-specific GOGAT is NADH-dependent. Be- cause both Fd-GOGAT and NADH-GOGAT are localized in plastids (Trepp et al., 1999), the novel NAD-MDH present in plastids of soybean may provide the NADH needed for the GOGAT reaction. Simultaneously, oxaloacetate is generated that may serve as an ammonium acceptor for the aspartate transaminase reaction.

Because the dry mass of a soybean seed is typically -6% N (Imsande, 1989), a soybean plant transports very large amounts of N to its developing seeds. Asparagine and gluta- mine are frequently the most abundant N-source found in the phloem entering developing seeds of many large-seeded le- gumes (Miflin and Lea, 1977; Pate, Peoples, and Atkins, 1984). In nodulated ureide producers such as soybean, allan- toin and allantoic acid also are usually present both in the xylem and the phloem. During reproductive growth, most of the ureide-N transported in the xylem to vegetative tissue is released as ammonia, whereupon it is re-assimilated, yielding asparagine and glutamine to be transported to the filling pods. Regardless of whether the phloem-borne-N that reaches the pods and developing seeds is an amide or ureide, much of the nitrogen is released as ammonia. Subsequently, rapid ammonia assimilation and amino acid synthesis is required to support protein synthesis. In turn, an abundant supply of 2-keto-a~- ceptor molecules, such as TCA-cycle intermediates oxaloace- tate and 2-oxoglutarate, are required for ammonia assimilation. Thus, the plastid-targeted soybean MDH, in conjunction with PEP carboxylase and other enzymes, might contribute directly to ammonia assimilation in pods and seeds (Ilarslan et al., 1997; Sakano, 1998) as well as in root nodules. Hence, it is proposed that the novel plastid-targeted NAD-MDH of soy- bean be considered a pod-enhanced MDH and a seed-enhanced MDH as well as a nodule-enhanced NAD malate de- hydrogenase.


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