High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification.

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.

Characterization of a soybean

Kunitz trypsin inhibitor, an abundant soybean [Glycine max (L.) Merr.] seed protein, has a molecular mass of 21500 Da and is specific for serine proteases. A soybean mutant (P.I. 196168) was characterized to determine the molecular basis for reduced Kunitz trypsin inhibitor levels during seed development. Western blot analysis revealed that P.I. 196168, in comparison to Amsoy 71, accumulated low amounts of Kunitz trypsin inhibitor protein. Non-denaturing polyacrylamide enzyme activity gels indicated that Amsoy 71 seeds contained at least five distinct zones of trypsin inhibitor activity. However, P.I. 196168 contained only four zones of enzyme inhibition. The coding region of the most abundant trypsin inhibitor gene (KTi3) was isolated from Amsoy 71 and P.I. 196168 by PCR. DNA sequence comparisons of the Kunitz trypsin inhibitor coding regions revealed two deletions and one G to T transversion have occurred. These mutations introduced four stop codons in the reading frame, resulting in a truncated protein. Northern blot analysis revealed that P.I. 196168 accumulated drastically lower amounts of KTi3 mRNA when compared with Amsoy 71.

A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.).

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.

Sinorhizobium fredii USDA257, a cultivar-specific soybean symbiont, carries two copies of y4yA and y4yB, two open reading frames that are located in a region that encodes the type III protein secretion system.

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean (Glycine max) cultivar Peking but fails to nodulate the improved cultivar McCall. Cultivar specificity is governed by a plasmid-borne locus, nolXBTUV. By DNA sequence analysis, we have identified two open reading frames, y4yA and y4yB, immediately downstream of nolX. Northern (RNA) blot analysis indicated that the expression of both y4yA and y4yB is inducible by isoflavonoids, and an intact copy of nolX is required. Two copies each of y4yA and y4yB are present in S. fredii USDA257, one on the sym plasmid (y4yAsp and y4yBsp), and the other on the chromosome (y4yAc and y4yBc). The cultivar-nonspecific strain USDA191 lacks y4yAc and y4yBc. Introduction of y4yAc plus y4yBc from USDA257 into USDA191 did not influence the ability of the latter strain to nodulate McCall soybean plants. Unlike nolX, the inactivation of y4yAsp and y4yBsp of USDA257 did not extend the host range of this strain. A double mutant, in which both the plasmid and chromosomal copies of y4yA and y4yB were mutated, had no observable effect on symbiotic ability of USDA257. The y4yAsp and y4yBsp mutants did not influence flavonoid-dependent extracellular protein production. Rhizobium sp. strain NGR234 and S. saheli USDA4893 both contain sequences similar to S. fredii USDA257 y4yAsp and y4yBsp; however, Bradyrhizobium spp., the traditional soybean symbionts, lack these genes.

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans.

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

Infection by Heterodera glycines Elevates Isoflavonoid Production and Influences Soybean Nodulation.

High-performance liquid chromatography and Sinorhizobium fredii USDA191 nodC-lacZ gene fusion were used to monitor changes in the isoflavonoid content of soybean roots infected with Heterodera glycines isolate TN1. Isoflavonoid concentrations in infected roots of both H. glycines-resistant Hartwig and susceptible Essex soybean were two to four-fold higher than those of uninfected roots 2 and 3 days after inoculation. The isoflavonoids produced activated the transcription of nodC-lacZ fusion. The most abundant isoflavonoids were identified as daidzein and genistein by HPLC and GC/MS. Heterodera glycines increased the number of nodules formed on Essex roots inoculated with B. japonicum (USDA110) but reduced shoot weight and decreased the net nitrogenase activity of the nodules. Heterodera glycines infection of resistant Hartwig did not affect the total number of nodules or their nitrogen- fixing capacity.

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli.

Rahnella aquatilis is a Gram-negative bacterium that can fix atmospheric nitrogen and also has the ability to solubilize mineral phosphate. We have cloned the genes that confer the mineral phosphate solubilizing (Mps) trait from this organism by mobilizing a cosmid library of R. aquatilis into Escherichia coli HB101. A 7.0-kb EcoRI fragment from a cosmid, when transferred into E. coli strains HB101 and DH5 alpha, conferred the ability to solubilize hydroxyapatite and the production of gluconic acid to E. coli. The relative amounts of soluble phosphate and gluconic acid produced by the cloned 7.0-kb EcoRI fragment in E. coli were significantly higher than those of R. aquatilis. Nucleotide sequence analysis revealed two complete open reading frames (ORF1 and ORF2) and a partial ORF, ORF1 and ORF2 encoded proteins of molecular mass 10 kDa and 44 kDa. The 44-kDa protein showed extensive sequence similarity to pqqE of Erwinia herbicola, Klebsiella pneumoniae and A. Acinetobacter calcoaceticus. The 10-kDa protein revealed strong similarity to the pqqD of K. pneumoniae and A. calcoaceticus.

The soybean cultivar specificity gene nolX is present, expressed in a nodD-dependent manner, and of symbiotic significance in cultivar-nonspecific strains of Rhizobium (Sinorhizobium) fredii.

Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner. This process is governed by a set of negatively acting nodulation genes termed nolXWBTUV. These genes prevent R. fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking. R. fredii strain USDA191 contains DNA sequences that hybridize to nolXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean. These sequences were isolated and their structure and function examined in comparison to nolXWBTUV of strain USDA257. Restriction maps of the two loci are identical, as is a 2-4 kb DNA sequence that corresponds to nolX and its promoter region. Expression of nolX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules. The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant. nolX is also present and flavonoid-inducible in HH103, a second R. fredii strain that nodulates McCall soybean normally. Inactivation of nolX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina. nolX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257.

ISRf1, a transposable insertion sequence from Sinorhizobium fredii.

Sinorhizobium fredii strain HH103, a nitrogen-fixing bacterial symbiont of plants, contains an insertion sequence (IS) that can transpose into plasmid pMUS248 and activate a promoterless TcR gene that is normally not expressed. We have cloned and characterized this element, which we designate ISRf1. The IS is 1002 bp in length, contains a single 513-bp open reading frame (ORF), is flanked by imperfect 36-bp terminal inverted repeats, and creates 5-bp target duplications. Two copies of ISRf1 are present in the genome of HH103, but it is absent from 12 other Sinorhizobium and Rhizobium strains. The element transposes at a frequency of 2.7 x 10(-6) per generation per cell.

Rhizobium fredii synthesizes an array of lipooligosaccharides, including a novel compound with glucose inserted into the backbone of the molecule.

Flavonoid cues from the plant host cause symbiotic, nitrogen-fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1). We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USADA191 and 257DH4 both produce a novel LCO with glucose substituted for N-acetyl-D-glucosamine in the backbone of the molecule.

Proteins from cells of Rhizobium fredii bind to DNA sequences precedingnolX, a flavonoid-inducible nod gene that is not associated with a nod box.

Rhizobium fredii strains USDA257 and USDA191 both contain a set of nodulation genes termed nolXWBTUV. In the USDA257 background, nolX prevents infection of soybean cultivars such as McCall, and in both backgrounds, it blocks nodulation of Erythrina spp. We report here that expression of nolX is differentially responsive to a panel of flavonoids, and that the most potent inducers are also the most active inducers of nodC, a conventional, nod box-associated gene. Cell-free protein extracts from uninduced and flavonoid-induced cells of strains USDA191 and USDA257 retard the electrophoretic mobility of DNA sequences that lie upstream of nolX. Binding is dependent both on nodD1 and nodD2, and it is abolished by the presence of a double-stranded, 23-bp oligonucleotide that lies within a 114-bp TaqI/SacII restriction fragment. This oligomer has significant sequence homology to A3, a putative negative regulatory element from R.leguminosarum bv. viciae. Deletion of the A3-homologous sequences elevates the basal and flavonoid-inducible expression of nolX by about 50%.

Morphometric Analysis of Rice Seed Protein Bodies (Implication for a Significant Contribution of Prolamine to the Total Protein Content of Rice Endosperm).

Electron microscopic observation of thin sections of rice (Oryza sativa L.) endosperm revealed two types of protein bodies (PBs): spherical and irregular-shaped ones. Immunocytochemical localization studies using antibodies raised against purified glutelins, prolamines, and globulins indicated that the prolamines were localized in the spherical PB, whereas the irregular-shaped PB contained glutelins and globulins. We counted and measured the surface area and the relative volume of 2303 PBs randomly selected from two different developmental stages and from different locations within the endosperm. The ratio of spherical to irregular-shaped PBs was 1:1.6. Double-label immunogold electron microscopic localization indicated that the globulins represented about 18% of the surface area of the irregular-shaped PBs. Based on our morphometric analysis, we estimate the relative contribution of glutelin as 53%, that of prolamine as 35%, and that of globulin as 12% of the total seed protein.

A new root-nodulating symbiont of the tropical legume Sesbania, Rhizobium sp. SIN-1, is closely related to R. galegae, a species that nodulates temperate legumes.

Rhizobium sp. SIN-1, isolated in India from root nodules on the tropical legume Sesbania aculeata, also induces nitrogen-fixing nodules on roots of S. macrocarpa, S. speciosa, S. procumbens, S. punicea, S. rostrata, and Vigna unguiculata. Unlike Azorhizobium caulinodans, SIN-1 does not induce stem nodules on S. rostrata. The nodules induced by SIN-1 develop exclusively at the bases of secondary roots. Electron microscopic studies of mature nodule sections revealed rhizobia within intercellular spaces, indicating a 'crack entry' mechanism of root infection. SIN-1 is a fast-growing, acid-producing, salt-tolerant Rhizobium that utilizes a wide variety of carbon sources. The nodulation (nod) genes of this strain are located on a 300-MDa symbiosis (sym) plasmid. Fatty acid profile and sequence comparison of a 260-bp conserved region of the 16S rRNA gene demonstrated that SIN-1 is phylogenetically closely related to R. galegae, a species that nodulates temperate legumes.

Transcriptional organization and expression of noIXWBTUV, a locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257.

Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert- and orientation-specific expression of two genes, noIX and noIW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.

Nucleotide and primary sequences of a rice endosperm cDNA are extensively homologous to elongation factor 1 beta'.

A rice endosperm cDNA expression library prepared with poly (A+) RNA was screened with polyclonal antibodies raised against purified 16 kD globulin. One of the three positive antigen producing clones was completely sequenced. This clone was composed of 759 bp and contained a single open reading frame encoding 192 amino acids. The 3' untranslated region did not include a typical polyadenylation signal, AATAAA. The cDNA encodes a protein of molecular weight 21,064 with an isoelectric point of 4.3. The amino acid sequence of the rice seed cDNA shows extensive homology to elongation factor 1 beta' from several sources, including human and Artemia salina. The molecular weight of rice seed EF-1 beta' is smaller than the others, and it lacks a conserved phosphorylation site that has been implicated in regulating nucleotide exchange activity.

Structures of nodulation factors from the nitrogen-fixing soybean symbiont Rhizobium fredii USDA257.

We have isolated and characterized the extracellular Nod factors of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean [Glycine max (L.) Merr.] and several other legume species. These signals are produced upon exposure to the isoflavone genistein and consist of a series of substituted, beta 1,4-linked tri-, tetra-, and pentamers of N-acetylglucosamine. N-Vaccenic acid replaces acetate on the nonreducing residue, and the reducing residue contains alpha-linked 2-O-methylfucose on carbon 6. Small amounts of a fucose-containing tetramer also were present. The Nod factors elicit root-hair deformations on soybean and two other plants at concentrations ranging from 10(-6) to 10(-12) M.

Nod factors of Rhizobium are a key to the legume door.

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lipo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japonicum strain USDA110. Between 10(-7) M and 10(-6) M NodNGR factors permitted these NodABC- mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a nonhost. Detailed cytological investigations of V. unguiculata showed that the NodABC- mutant NGR delta nodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257.

Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolW and nolX are of the opposite polarity. The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.