A specialised proteomic database for comparing matrix-assisted laser desorption/ionization-time of flight mass spectrometry data of tryptic peptides with corresponding sequence database segments.

We have developed a specialised proteomic database for the analysis of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) data derived from tryptic peptides of Sinorhizobium meliloti proteins. This database currently contains the amino acid sequence data of the proteins predicted from the complete chromosome, MALDI-TOF MS data from proteolytic peptides of about 400 tryptically digested proteins, and the results of a search of the MALDI-TOF MS spectra against the chromosomal amino acid sequences. The database made it possible to access and compare the sequences of theoretical tryptic peptides that correspond to MALDI-TOF peaks in the mass spectrum with predicted tryptic peptides from identified proteins that could not be matched to MALDI-TOF peaks. A comparison of the molecular weights, isoelectric points and amino acid compositions of the identified and nonidentified peptides is presented. We also show how the system can assist in the development of an automated scoring function that facilitates and consolidates protein identification.

Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting.

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2,500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105,000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Characterisation of rice anther proteins expressed at the young microspore stage.

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.

Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development.

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in beta-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can "hijack" cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.

Identification of nolR-regulated proteins in Sinorhizobium meliloti using proteome analysis.

Extractable proteins from Sinorhizobium meliloti strains AK631 and EK698 (a Tn5-induced noIR-deficient mutant of AK631), grown in tryptone agar (TA) medium with or without the addition of the plant signal luteolin, were separated by two-dimensional gel electrophoresis and compared. Analysis of silver-stained gels showed that the noIR mutant had 189 proteins that were significantly altered in their levels (101 protein spots up- and 88 downregulated). Coomassie-stained preparative two-dimensional (2-D) gels or polyvinylidene difluoride (PVDF) membranes blotted from preparative gels showed that at least 52 of the altered proteins could be reproducibly detected and isolated from the noIR mutant. These 52 altered protein spots were classified into five groups based on an assessment of protein abundance by computer analysis and the effect of the presence or absence of luteolin addition to the growth medium. N-terminal microsequencing of 38 proteins revealed that the most striking feature of the consequence of the noIR mutation is the number and broad spectrum of cellular functions that are affected by the loss of the NoIR function. These include proteins involved in the tricarboxylic acid (TCA) cycle, heat shock and cold shock proteins, protein synthesis, a translation elongation factor, oxidative stress and cell growth and maintenance functions. We propose that the NoIR repressor is a global regulatory protein which responds to environmental factors to fine-tune intracellular metabolism.

Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives of Sinorhizobium meliloti strain 2011.

Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium. Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146). Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins. However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818. While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains. These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin. Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma. These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S. meliloti cells. Proteome analysis provides a sensitive tool to examine the functional organisation of the S. meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021.

Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development.

We report improved method for white clover (Trifolium repens) transformation using Agrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. The bar gene and phosphinothricin selection was shown to be a more effective selection system than nptII (kanamycin selection) or aadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (beta-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

Rhizobium inoculation and physical wounding result in the rapid induction of the same chalcone synthase copy in Trifolium subterraneum.

The gene or genes encoding chalcone synthase (CHS) in the legume Trifolium subterraneum (subterranean clover) were induced within 6 hr after inoculation with Rhizobium leguminosarum bv. trifolii strain ANU843. No induction was found in uninoculated controls or plants inoculated with either the nodulation-deficient R. l. bv. trifolii strain ANU845 (pSym-) or R. meliloti strain 1021, which is capable of nodulating alfalfa but not clover. Morphological examination of the interaction between the legume and bacteria in this system showed that root hair distortion (a marker of the early events in the interaction) was apparent within 10 hr after inoculation. This indicated that CHS induction could occur before any detectable sign of rhizobial penetration of root hairs. The addition of a crude preparation of R. l. bv. trifolii lipooligosaccharide signals (Nod metabolites) to the plant growth medium had no effect on the expression of CHS over 36 hr, although root hair distortion was apparent over this time. These treatments were then contrasted with physical wounding. Wounding the plants led to a rapid induction of CHS, occurring within 2 hr. Sequence analysis of cloned CHS cDNA from pools sampled after Rhizobium inoculation or wounding treatments showed the gene designated CHS5 was the major CHS species in both treatments. Conserved sequences were found in promoters of CHS5 and soybean Gmchs7, a gene which has overlapping expression patterns. These findings support the view that the induction of the phenylpropanoid pathway is involved in the very early events of the Rhizobium infection of legumes.

In Trifolium subterraneum, chalcone synthase is encoded by a multigene family.

Chalcone synthase (CHS) catalyzes the first and key regulatory step in flavonoid biosynthesis. We report the existence and characterization of a CHS multigene family present in Trifolium subterraneum L. cultivar Karridale. The CHS family consists of at least four members, which are tightly clustered in a 15-kb region. The complete sequences of two of these genes (CHS1 and CHS2) are presented. The putative promoters of these genes have sequences which are homologous to those known, or implicated, in regulation of the expression of phenylpropanoid-encoding genes.

Characterization of a phenylalanine ammonia-lyase multigene family in Trifolium subterraneum.

The enzyme phenylalanine ammonia-lyase (PAL) was found to be encoded by a small gene family in the legume Trifolium subterraneum (subterranean clover). At least three of the family members are tightly clustered within approx. 20 kb of DNA. Sequencing of one of the genes established that it possesses two exons, the position of the single intron being identical to that found for PAL genes from other plants. The PAL protein consists of 725 amino acids, as deduced from the nucleotide sequence.

Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules.

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.

Structural analysis of the genes encoding the molybdenum-iron protein of nitrogenase in the Parasponia rhizobium strain ANU289.

The genes encoding the Molybdenum-Iron protein component of nitrogenase (nifD and nifK) have been identified and fully characterised in the Parasponia Rhizobium strain ANU289. The two genes are contiguous and are separated from the gene encoding the Fe-protein component of nitrogenase (nifH) by 21 kb of DNA. We present the entire DNA sequence of the nifD and nifK genes, thus completing the characterisation of the primary structure of the nitrogenase genes in this Rhizobium strain. Comparison of the sequence preceding the transcription initiation point of nifDK with that preceding nifH reveals a consensus promoter sequence 5'-PyTGGCAPyG-4 bp-TTGC(T/A)-10 bp-3'. This consensus promoter is found preceding nif genes in both fast-growing and slow-growing Rhizobium strains and shows a structural similarity to that preceding the coordinately-regulated nif operons in the asymbiotic organism Klebsiella pneumoniae.