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Physiology and Biochemistry |
2Departments of Agronomy and of Zoology/Genetics, Iowa State University, Ames, Iowa 50011-1010 USA; 3Maltagen Forschung GmbH, Schaarstrasse 1, D-56626 Andernach, Germany; 4Pflanzenphysiologie, Fachbereich Biologie/Chemie, Universitat Osnabruck, D-49069, Germany; 5Technische Universität München, Lehrstuhl für Botanik, Biologikum-Weihenstephan, Am Hochanger 4, D-85350 Freising, Germany; and 6USDA-ARS-CICGR Unit and Departments of Agronomy and of Zoology/Genetics Iowa State University, Ames, Iowa 50011-1010 USA
Received for publication March 13, 2001. Accepted for publication June 19, 2001.
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ABSTRACT |
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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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Vance et al., 1994
; Miller et al., 1998
; Waters et al., 1998
; Fedorova, Tikhonovich, and Vance, 1999
; Trepp et al., 1999
). A nodule-enhanced phosphoenolpyruvate (PEP) carboxylase and a nodule-enhanced malate dehydrogenase (neMDH) in the nodule provide oxaloacetate (OAA) to support synthesis of aspartate and asparagine and the continuation of the tricarboxylic acid (TCA) cycle. Because N can account for 6% of the dry mass of a soybean seed (Imsande, 1989
), we reasoned that grain legume seed formation may require enhanced activities of PEP carboxylase and MDH to support rapid synthesis of amino acids and seed storage proteins. Indeed, the soybean genome encodes at least four PEP carboxylases, two of which are expressed in the seed (Hata, Izui, and Kouchi, 1998
). Furthermore, in Vicia faba, enhanced nitrogen availability can induce the mRNAs for both a PEP carboxylase and legumin B, a major seed storage protein (Weber et al., 1998
; Golombek et al., 1999
). Also, an active MDH, whose function is similar to the neMDH found in alfalfa (Miller et al., 1998
) and pea nodules (Fedorova, Tikhonovich, and Vance, 1999
), would be required to facilitate OAA synthesis and metabolism during soybean seed filling. Malate dehydrogenase would catalyze the reduction of OAA to malate, thereby increasing net OAA synthesis from PEP and providing malate to support the TCA cycle, fatty acid biosynthesis, and other cellular activities (Plaxton, 1996
; Golombek et al., 1999
). These latter reactions are important because the seed-enhanced PEP carboxylase is strongly activated by glucose-6-phosphate, whereas aspartate, glutamate, and malate are inhibitors of the enzyme (Golombek et al., 1999
). In addition, oxalate is known to accumulate in developing soybean seeds (Ilarslan et al., 1997
). Oxalate is a potent inhibitor of pyruvate kinase, an enzyme that competes directly with PEP carboxylase for the utilization of PEP (Smith, Knowles, and Plaxton, 2000
). Coordination of these various metabolic activities is under stringent metabolic control and helps regulate the availability of precursor molecules needed for protein synthesis and seed maturation (Plaxton, 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, glyoxysomes, peroxisomes, mitochondria, and apparently the chloroplast (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 dehydrogenase (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 tissue.
The broad objectives of our studies were to (1) clone and sequence several of the MDH genes present in soybean (Imsande 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 subunit, 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. Because 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) was double-digested with EcoRI and XbaI and cloned into the EcoRI-site of the lambdaZAPII-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 mitochondrial malate dehydrogenase cDNA clone (Gietl, Lehnerer, and Olsen, 1990
). Positive phage clones were converted into the plasmid vector pBluescript SK by in vivo-excision according to the manufacturer's instructions and sequenced. In addition to several clones coding for mitochondrial 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 neMDH. 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 hydrogenase, the 530 bp (base pair) region of the 4.8-kb XbaI fragment that contained 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 screening 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 (Stratagene). A 5.5-kb DNA fragment was sequenced and found to contain the complete 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 sequencing, pNEMDH8 was used for coupled in vitro transcription/translation using TNT Coupled reticulocyte Lysate System (Promega, Heidelberg, Germany) 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 recollected and fractionated into the envelope membranes and thylakoids and stroma according to Flügge et al. (1989)
. The samples were subsequently analyzed 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 extract, various soybean tissues were ground in liquid nitrogen and extracted with 1 mL of extraction buffer (20 mmol/L 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 homogenate was incubated on ice for 15 min and then centrifuged at 20 000 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 µg 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) 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 successfully 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 fragment. Hence, it is unlikely that the complete ggh gene has been isolated. The putative ggh protein encoded by the 5.5-kb fragment was composed of 462 amino acids, 50 of which constitute an apparent transit peptide (GBAN-AAD28640). The mature putative protein encoded by the 5.5 kb fragment (i.e., amino acid residues 51–462) was 91% identical and 95% similar to the putative ggh protein from tobacco (GBAN-CAA07683), 87% identical and 94% similar to the ggh from Arabidopsis (GBAN-CAA74372; Keller et al., 1998
), 87% identical and 92% similar to the ggh from Mesembryanthemum crystallinum (GBAN-AAC19396), and 68% identical and 79% similar to the ggh produced by Synechocystis (GBAN-CAA66615; Addlesee et al., 1996
) (Fig. 1). Although the putative transit peptides for the geranylgeranyl hydrogenases from Arabidopsis, tobacco, and soybean were all 44–50 amino acids residues in length and rich in serine, their sequence similarities were generally <50%.
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95 amino acids, whereas the mature protein would contain 317 amino acids residues (GBAN-AAC24855). The mature MDH protein was 92% identical and 96% similar to the putative neMDH from Pisum sativum (GBAN-AAC28106), 91% identical and 96% similar to the neMDH from Medicago sativa (GBAN-AAB99757), 87% identical and 94% similar to the chloroplastic NAD-MDH from Arabidopsis (GBAN-CAA74320; Berkemeyer, Scheibe, and Ocheretina, 1998
), and 65% identical and 78% similar to the glyoxysomal MDH from soybean (GBAN-P37228; Guex et al., 1995
). Only two transit peptides found in GenBank showed similarity to that of the novel MDH described in this report. The 95-amino acid transit peptide of the novel MDH was 64% identical and 72% similar to that of the neMDH from Medicago sativa and 53% identical and 58% similar to that of the neMDH from Pisum sativum (GBAN-AAC28106) (Fig. 2). On the other hand, there was little sequence similarity between the transit peptide of the chloroplastidic NAD-MDH and the novel MDH.
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45 kDa (Fig. 4). Isolated intact spinach chloroplasts were incubated with the translation mixture either in the light and in the presence of external ATP or in the dark without ATP. After external protease treatment and subsequent fractionation of the chloroplasts into stroma, thylakoids, and envelope membranes, a labeled polypeptide of the size of the mature product (35 kDa) was detected exclusively in the stroma fraction of illuminated chloroplasts (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The mRNA was expressed in E. coli, and some of the properties of this multifunctional enzyme were examined. Keller et al. (1998)
concluded that the multifunctional enzyme catalyzes the reduction of geranylgeranyl-chlorophyll a to phytyl-chlorophyll a as well as the reduction of free geranylgeranyl diphosphate to phytl diphosphate. The amino acid sequence of the putative geranylgeranyl hydrogenase encoded by the soybean genomic fragment described in this report is 87% identical and 94% similar to that of the putative sequence encoded by the Arabidopsis cDNA clone. Likewise, there is 91% identity and 95% similarity to the amino acid sequence reported for ggh from tobacco (Fig. 1). With the exception of a valine-for-isoleucine replacement, there is complete identity within the 24-bp nucleotide binding domain of the enzymes from tobacco and soybean. Thus, based on putative amino acid sequence analysis, we report the cloning and sequencing of a soybean geranylgeranyl hydrogenase. Although the amino acid sequences of the putative transit peptides are similar, they are not as highly conserved as those of the mature proteins.
Characterization of a plastidic MDH from soybean
A plastid-targeted soybean MDH is located 1680 bp downstream from the UGA stop codon of ggh. Although the deduced amino 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-AAB99757; 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 ammonia 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 glycinamide ribonucleotide, a second molecule of glutamine for the synthesis of formylglycinamidine ribonucleotide, aspartate for the synthesis of aminoimidazole ribonucleotide, and two molecules 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 isoforms 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. Because 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 glutamine are frequently the most abundant N-source found in the phloem entering developing seeds of many large-seeded legumes (Miflin and Lea, 1977
; Pate, Peoples, and Atkins, 1984
). In nodulated ureide producers such as soybean, allantoin 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-acceptor molecules, such as TCA-cycle intermediates oxaloacetate 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 soybean be considered a pod-enhanced MDH and a seed-enhanced MDH as well as a nodule-enhanced NAD malate dehydrogenase.
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FOOTNOTES |
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7 Author for reprint requests (telephone: 515-294-7378; FAX: 515-294-2299; rpalmer{at}iastate.edu
).
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LITERATURE CITED |
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