Decreased NADH glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with an antisense glutamate synthase transgene.

Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N2 fixation in root nodules. The glutamine synthetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) cycle is responsible for the initial N assimilation. This report describes the analysis of a transgenic alfalfa (Medicago sativa L.) line containing an antisense NADH-GOGAT (EC 1.4.1.14) under the control of the nodule-enhanced aspartate amino-transferase (AAT-2) promoter. In one transgenic line, NADH-GOGAT enzyme activity was reduced to approximately 50%, with a corresponding reduction in protein and mRNA. The transcript abundance for cytosolic GS, ferredoxin-dependent GOGAT (EC 1.4.7.1), AAT-2 (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were unaffected, as were enzyme activities for AAT, PEPC and GS. Antisense NADH-GOGAT plants grown under symbiotic conditions were moderately chlorotic and reduced in growth and N content, even though symbiotic N2 fixation was not significantly reduced. The addition of nitrate relieved the chlorosis and restored growth and N content. Surprisingly, the antisense NADH-GOGAT plants were male sterile resulting from inviable pollen. A reduction in NADH-GOGAT enzyme activity and transcript abundance in the antisense plants was measured during the early stages of flower development. Inheritance of the transgene was stable and resulted in progeny with a range of NADH-GOGAT activity. These data indicate that NADH-GOGAT plays a critical role in the assimilation of symbiotically fixed N and during pollen development.

Subnuclear distribution of the entire complement of linker histone variants in Arabidopsis thaliana.

Linker histones (e.g. H1, H5, H1 degrees ) are thought to exert control on chromatin function by restricting nucleosomal dynamics. All higher eukaryotes possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complement of linker histone structural variants and therefore is an ideal model for investigating functional differentiation among linker histones. Histones H1-1 and H1-2 are relatively similar proteins that are expressed in a wide variety of tissues and make up the majority of linker histone while H1-3 is a highly divergent minor variant protein that is induced by drought stress. We are interested in determining whether the in vivo distribution of each of these proteins also differs. To this end, we have produced subtype-specific antibodies and have localized each of the three proteins at the intranuclear and DNA sequence levels by indirect immunofluorescence and immunoprecipitation, respectively. Antibodies against linker histones H1-1 and H1-2 decorate nuclei in patterns very similar to 4',6-diamidino-2-phenylindole (DAPI) staining, but different than the staining pattern of total histones. In contrast, antibodies made against two regions of H1-3 bind to chromatin in a diffuse pattern distinct from the DAPI-staining pattern. We also describe a technique to determine the localization of plant linker histone variants along regions of chromatin, employing in vivo chemical DNA-protein cross-linking to preserve native associations followed by immunoprecipitation with subtype-specific antibodies. We use this technique to demonstrate that, in contrast to the major linker histones, H1-3 does not bind the repetitive sequences pAL1 and 5S rDNA. In addition, we show that linker histones are bound to the compacted nucleosomal arrays at the telomere but with reduced stoichiometry. Taken together, our results suggest that plants, as has been shown for animals, possess a variant linker histone that is differentially localized.

Molecular genetic analysis of the drought-inducible linker histone variant in Arabidopsis thaliana.

Linker histones are ubiquitous structural components of chromatin that have been shown to influence the expression of a subset of genes in diverse organisms. Plants contain a minor linker histone variant that is expressed in most tissues of all organs, and is induced during drought stress. Based on reporter gene analysis in roots, His1-3 is expressed almost exclusively in emerging secondary roots in unstressed plants, but is primarily expressed in the root meristem and elongation zone of stressed plants. In shoots, expression is higher in younger tissues than older tissues. In order to investigate the function of H1-3, we have generated lines with altered levels of H1-3. Plants expressing an antisense His1-3 transcript exhibit a greatly impaired induction (5% of wild-type RNA levels during stress) of His1-3 transcripts in shoots during drought and contain decreased protein relative to wild-type control plants. In plants overexpressing His1-3, more H1-3 is bound to chromatin than in unstressed wild-type plants. None of the plants containing these transgenes display phenotypic aberrations or differences in water content during drought stress. Additionally, the expression of several drought-responsive genes is not significantly altered in lines misexpressing His1-3.

The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog.

Development of root nodules, specifically induction of cortical cell division for nodule initiation, requires expression of specific genes in the host and microsymbiont. A full-length cDNA clone and the corresponding genomic clone encoding a MAP (mitogen-activated protein) kinase homolog were isolated from alfalfa (Medicago sativa). The genomic clone, TDY1, encodes a 68.9-kDa protein with 47.7% identity to MMK4, a previously characterized MAP kinase homolog from alfalfa. TDY1 is unique among the known plant MAP kinases, primarily due to a 230 amino acid C-terminal domain. The putative activation motif, Thr-Asp-Tyr (TDY), also differs from the previously reported Thr-Glu-Tyr (TEY) motif in plant MAP kinases. TDY1 messages were found predominantly in root nodules, roots, and root tips. Transgenic alfalfa and Medicago truncatula containing a chimeric gene consisting of 1.8 kbp of 5' flanking sequence of the TDY1 gene fused to the beta-glucuronidase (GUS) coding sequence exhibited GUS expression primarily in the nodule parenchyma, meristem, and vascular bundles, root tips, and root vascular bundles. Stem internodes stained intensely in cortical parenchyma, cambial cells, and primary xylem. GUS activity was observed in leaf mesophyll surrounding areas of mechanical wounding and pathogen invasion. The promoter was also active in root tips and apical meristems of transgenic tobacco. Expression patterns suggest a possible role for TDY1 in initiation and development of nodules and roots, and in localized responses to wounding.

NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression.

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH.

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here, we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene is comprised of 13 exons separated by 12 introns. The 5' flanking region of the AS gene confers nodule-enhanced reporter gene activity in transformed alfalfa. This region also confers enhanced reporter gene activity in dark-treated leaves. These results indicate that the 5' upstream region of the AS gene contains elements that affect expression in root nodules and leaves. Both AS mRNA and enzyme activity increased approximately 10- to 20-fold during the development of effective nodules. Ineffective nodules have strikingly reduced amounts of AS transcript. Alfalfa leaves have quite low levels of AS mRNA and protein; however, exposure to darkness resulted in a considerable increase in both. In situ hybridization with effective nodules and beta-glucuronidase staining of nodules from transgenic plants showed that AS is expressed in both infected and uninfected cells of the nodule symbiotic zone and in the nodule parenchyma. RNA gel blot analysis and in situ hybridization results are consistent with the hypothesis that initial AS expression in nodules is independent of nitrogenase activity.

Analyses of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a crucial role in the assimilation of CO2 during symbiotic N2 fixation in legume root nodules. In this study, an alfalfa PEPC gene (PEPC-7), whose transcripts are found at elevated levels in nodules relative to either leaves or roots, has been isolated and characterized. The intron/exon structure of this gene is identical to that of most other plant PEPC genes except for the presence of an additional intron in the 5' untranslated region. In situ RNA hybridization studies showed that PEPC transcripts were present in the nodule meristem, the infection zone, the nitrogen-fixing zone, and the parenchyma. PEPC transcripts were also found in vascular tissue of roots and nodules and in the pulvinus of petioles. In transgenic alfalfa, a chimeric reporter gene was expressed in these same regions except that little expression was found in the nodule meristem. Analyses of promoter deletions suggest that the region between -634 and -536 is of particular importance in directing transcriptional activity to the infected zone of nodules. Within this region is a mirror repeat sequence that is potentially capable of forming an H-DNA structure. These results indicate that PEPC-7 has a central role in nitrogen-fixing nodules and that regulation of transcription is an important determinant of its activity.

A drought-stress-inducible histone gene in Arabidopsis thaliana is a member of a distinct class of plant linker histone variants.

We have isolated and characterized a gene, His1-3, encoding a structurally divergent linker histone in Arabidopsis thaliana. Southern and northern hybridization data indicate that A. thaliana expresses three single-copy linker histone genes, each encoding a structurally distinct variant. H1-3 is a considerably smaller protein (167 amino acids with a mass of 19.0 kDa) than any other described linker histone from higher eukaryotes. We examined the expression of His1-3 at the RNA and protein levels and found that it is induced specifically by water stress. In contrast, expression of His1-1, His1-2 and His4 appear unaffected by water stress. Furthermore, the primary structure of the protein possesses distinct characteristics that are shared with another drought-inducible linker histone, H1-D, isolated from Lycopersicon pennellii. Based on structural characteristics of the deduced protein and its inducible expression, we hypothesize that H1-3 and H1-D are linker histone variants that have specialized roles in the structure and function of plant chromatin and therefore they can be considered to be members of a unique subclass of plant histones. Immunoblotting with an antibody produced against a short polypeptide in the conserved domain of this subtype indicates that similar proteins may exist in other plants.

Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation.

Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.

Chlamydomonas transcripts encoding three divergent plastid chaperonins are heat-inducible.

Three cDNAs encoding plastid cpn60 chaperonin subunits have been isolated from the unicellular green alga Chlamydomonas reinhardtii. Based on comparisons of the predicted amino acid sequences, we conclude that Chlamydomonas, like higher plants, contains divergent plastid cpn60-alpha and cpn60-beta subunits. The predicted amino acid sequences of the two Chlamydomonas cpn60-beta subunits differ significantly (24% divergent), indicating that the two cpn60-beta subunits have been selectively maintained for a considerable period of time. Unlike plastid chaperonin transcripts in higher plants, heat shock conditions (42 degrees C) lead to a rapid increase (10- to 30-fold) in the level of each of the three plastid transcripts.

Genomic structure, expression and evolution of the alfalfa aspartate aminotransferase genes.

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.

The Arabidopsis thaliana ribosomal protein S15 (rig) gene.

We have isolated cDNA and genomic clones for Arabidopsis thaliana cytosolic ribosomal protein S15 and determined their sequences. Like animal S15 genes, this plant S15 gene is composed of four exons and the first intron is located immediately following the ATG translational start codon. The 5' end of the S15 mRNA was mapped by RNase protection experiments which showed that this mRNA contains a 5' untranslated region of approx. 83 nucleotides. Southern blot analyses suggest that Arabidopsis S15 is encoded by a small family of genes. The sequences of the predicted exons in the cloned S15 gene are identical to that of the S15 cDNA, demonstrating that this gene is transcriptionally active. Sequence analysis of the cloned A. thaliana S15 gene shows that it is tightly linked (approx. 500 nucleotides distant) to a gene of unknown function. The Arabidopsis S15 protein described here is about 75% identical to vertebrate S15, 70% identical to the homologous yeast protein (S21), 50% identical to archaebacterial S19, 30% identical to eubacterial S19, and about 30% identical to plant mitochondrial and plastid S19.

Molecular analysis of allelic polymorphism at the AAT2 locus of alfalfa.

Aspartate aminotransferase (AAT) plays a key enzymatic role in the assimilation of symbiotically fixed nitrogen in legume root nodules. In alfalfa, two distinct genetic loci encode dimeric AAT enzymes: AAT1, which predominates in roots, and AAT2, which is expressed at high levels in nodules. Three allozymes of AAT2 (AAT2a, -2b and -2c), differing in net charge, result from the expression of two alleles, AAT2A and AAT2C, at this locus. Utilizing antiserum to alfalfa AAT2, we have previously isolated from an expression library one AAT2 cDNA clone. This clone was used as a hybridization probe to screen cDNA libraries for additional AAT2 cDNAs. Four different clones were obtained, two each that encode the AAT2a and AAT2c enzyme subunits. These two sets of cDNAs encode polypeptides that differ in net charge depending upon the amino acid at position 296 (valine or glutamic acid). Within each set of alleles, the two members differ from each other by the presence or absence of a 30 bp (ten amino acid) sequence. The presence or absence of this ten amino acid sequence has no effect on the size or charge of the mature AAT2 protein because it is located within the region encoding the protein's transit peptide, which is proteolytically removed upon transport into plastids. The data suggest that a deletion event has occurred independently in two AAT2 progenitor alleles, resulting in the four allelic cDNA variants observed. The deletion of this ten amino acid sequence does not appear to impair the normal maturation of the enzyme.

Differential expression of six glutamine synthetase genes in Zea mays.

The maize genome has been shown to contain six glutamine synthetase (GS) genes with at least four different expression patterns. Noncoding 3' gene-specific probes were constructed from all six GS cDNA clones and used to examine transcript levels in selected organs by RNA gel blot hybridization experiments. The transcript of the single putative chloroplastic GS2 gene was found to accumulate primarily in green tissues, whereas the transcripts of the five putative GS1 genes were shown to accumulate preferentially in roots. The specific patterns of transcript accumulation were quite distinct for the five GS1 genes, with the exception of two closely related genes.

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N2 fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.

Characterization of rps17, rp19 and rpl15: three nucleus-encoded plastid ribosomal protein genes.

Approximately two-thirds of the 55 to 60 plastid ribosomal proteins are encoded in the nucleus. Since the protein products of each of these genes are needed in equal amounts for ribosome assembly, their expression may be coordinately regulated by common mechanisms. To begin to understand how the expression of these genes is regulated, we have isolated cDNA and genomic clones for three plastid ribosomal protein genes from an Arabidopsis thaliana library. The genes rps17, rpl9 and rpl15, encoding plastid ribosomal proteins CS17, CL9 and CL15, respectively, are located in the nuclear genome and Southern blot data suggest that each is a single copy gene in A. thaliana. Northern blot data show that transcripts from rps17, rpl9 and rpl15 are much more abundant in leaves and stems than they are in roots. The nucleotide sequences of each of these three genes were determined and their transcriptional initiation sites identified. rps17 transcripts have multiple 5' ends suggesting that they are initiated at multiple sites or are post-transcriptionally processed at their 5' end. rpl9 and rpl15 apparently have unique transcriptional initiation sites but are post-transcriptionally processed to remove six and three introns, respectively, from their primary transcripts. We have examined the genomic sequences for motifs that may be important for the proper expression of these genes. A 7 bp sequence motif, whose consensus is 5'-AGGCCCA-3', flanked by AT-rich regions was identified between 38 and 73 nucleotides upstream of the rps17, rpl9 and rpl15 transcriptional initiation sites.

Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels.

Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C(4) species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT-2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness.