BackgroundMembers of the legume genus Lupinus exude phloem 'spontaneously' from incisions made to the vasculature. This feature was exploited to document macromolecules present in exudate of white lupin (Lupinus albus [L.] cv Kiev mutant), in particular to identify proteins and RNA molecules, including microRNA (miRNA).ResultsProteomic analysis tentatively identified 86 proteins from 130 spots collected from 2D gels analysed by partial amino acid sequence determination using MS/MS. Analysis of a cDNA library constructed from exudate identified 609 unique transcripts. Both proteins and transcripts were classified into functional groups. The largest group of proteins comprised those involved in metabolism (24%), followed by protein modification/turnover (9%), redox regulation (8%), cell structural components (6%), stress and defence response (6%) with fewer in other groups. More prominent proteins were cyclophilin, ubiquitin, a glycine-rich RNA-binding protein, a group of proteins that comprise a glutathione/ascorbate-based mechanism to scavenge oxygen radicals, enzymes of glycolysis and other metabolism including methionine and ethylene synthesis. Potential signalling macromolecules such as transcripts encoding proteins mediating calcium level and the Flowering locus T (FT) protein were also identified. From around 330 small RNA clones (18-25 nt) 12 were identified as probable miRNAs by homology with those from other species. miRNA composition of exudate varied with site of collection (e.g. upward versus downward translocation streams) and nutrition (e.g. phosphorus level).ConclusionsThis is the first inventory of macromolecule composition of phloem exudate from a species in the Fabaceae, providing a basis to identify systemic signalling macromolecules with potential roles in regulating development, growth and stress response of legumes.

In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery -possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.

The HLA-B locus is extremely polymorphic. We have sequenced a region, CL, telomeric of HLA-B that also shows a high degree of allelic variation which we have shown previously by RFLP analysis. The polymorphism can be accounted for by sequence variation in duplicated, reiterated sequence elements called geometric elements. Comparison of the CL1 and CL2 sequences from the 57.1, 8.1, 18.2 and 7.1 ancestral haplotypes revealed that the lengths of the elements vary, both between the duplicated loci within a haplotype and between haplotypes, apparently because certain sequences are inserted or deleted. It is possible, using the polymerase chain reaction, to amplify these elements in genomic DNA from ancestral haplotypes for which sequence data of the CL region are not available and to obtain gel patterns which are characteristic of different ancestral haplotypes. The most striking feature of the data is the fact that the majority of the CL patterns are haplospecific; i.e. have a particular pattern that is unique for a particular ancestral haplotype and can be used to type these ancestral haplotypes. At least 12 different allelic patterns have been identified within a panel of 29 cell lines representing 16 ancestral haplotypes. For these 16 ancestral haplotypes, all examples of each haplotype have the same CL pattern. The haplotypic nature of the patterns confirms that ancestral haplotypes are conserved chromosomal segments and that coding and non-coding sequences are identical by descent from a remote ancestor.

De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. 35 S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATPdependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.In root nodules of legumes of the tribe Phaseoleae, symbiotically fixed N 2 is assimilated by de novo purine biosynthesis, after which the purines are oxidized to allantoin and allantoic acid (Atkins and Smith, 2000). It is in this form that fixed N is exported in xylem from the nodule to the shoot. The 10 enzymes of the purine biosynthetic pathway have been well studied in relation to their role in cancer metabolism, and the genes encoding them cloned from a wide variety of organisms. However, in plants, far less is known about the enzymes of the pathway, although genes encoding a number have now been isolated (Atkins and Smith, 2000).The cDNA sequences of all the plant purine biosynthetic (pur) genes studied have 5Ј extensions relative to the coding regions of the corresponding Escherichia coli genes (Senecoff and Meagher, 1993;Chapman et al., 1994;Schnorr et al., 1994Schnorr et al., , 1996Kim et al., 1995;Senecoff et al., 1996;Smith et al., 1998). These extensions potentially encode N-terminal, organelle-targeting sequences and indicate that in contrast to other eukaryotes (Gooljarsingh et al., 2001), the pathway is likely to be organelle-localized in plants. This was confirmed in a recent study that showed that in cowpea (Vigna unguiculata L. Walp) nodules, both the mitochondria ...