Polyphyletism of Celastrales deduced from a chloroplast noncoding DNA region.

In a previous study we examined the phylogeny of four families related to the angiosperm order Celastrales based on chloroplast rbcL 5' flanking sequences. We have added here several additional dicots, sampled from 6 of the 7 families of Celastrales sensu Cronquist and 19 putatively related genera. Based on a cladistic analysis of these DNA sequences, the order Celastrales appears polyphyletic: it is here restricted to Celastraceae (including Hippocrateaceae and Brexia) with Parnassia as sister; Aquifoliaceae plus Helwingia are included in Asteridae. Neither Salvadoraceae nor Geissolomataceae, Icacinaceae, Phellinaceae, Aextoxicaceae, Corynocarpaceae, Dichapetalaceae, Stackhousiaceae, or Goupiaceae are related to Celastrales. The usefulness of this noncoding region is discussed and the influence of the A + T content of neighboring bases on the increase of transversions is also observed as previously shown in chloroplast noncoding regions of monocots.

Comparison of the evolution of ribulose-1, 5-biphosphate carboxylase (rbcL) and atpB-rbcL noncoding spacer sequences in a recent plant group, the tribe Rubieae (Rubiaceae).

Plastid sequences of the atpB-rbcL spacer and rbcL gene itself were used to evaluate their respective potential in reconstructing the phylogeny of 15 taxa from the tribe Rubieae (Rubiaceae). From our previous analyses using the atpB-rbcL spacer, the 15 selected taxa represent most of the variability of the tribe. Since this group is considered to be relatively recent (Upper Tertiary), it should allow the study of early dynamics of nucleotide substitutions in recent divergences. The results show that the spacer and rbcL inferred phylogenies are not totally congruent; the spacer trees are more similar to interpretations of morphological data. A comparative analysis of the pattern of nucleotide substitution of these two sequences in the Rubieae shows that (1) the overall rate of substitution is similar in the spacer and in rbcL, and the rate of synonymous substitution in rbcL is much higher; (2) the level of homoplasy is higher in rbcL than in the spacer matrix which shows a higher phylogenetic structure; and (3) the pattern of transition and transversion substitutions is different in the two sequences, and is not linear in rbcL. As a result of these observations, we suggest that (1) the spacer is evolving relatively slowly because of unsuspected, and phylogenetically important, selective constraints on its sequence; and (2) in the rbcL sequence, many sites, free of constraint, are changing at high rate, and some of these sites seem to have undergone multiple substitutions even in this recent tribe. This could explain the high level of homoplasy found in Rubieae rbcL sequences.

Chloroplast DNA variation and parentage analysis in 55 apples.

The chloroplastic atpB-rbcL spacer and the first 53 codons of the rbcL coding sequence was sequenced for 40 apple cultivars and 15 wild species. This chloroplast DNA region is 904 base pairs long, and only five mutations sites were found among the tested samples. Although the cpDNA variation was low, some parentages are proposed based on the maternal inheritance of plastid DNA: the male and female parents are specified, or else suggested, for Worcester, Discovery, Starking, Starkrimson, Kidd's Orange Red, Priscilla, and Gloster, as well as for the putative wild origin for Malus x domestica.

The atpB and rbcL promoters in plastid DNAs of a wide dicot range.

The plastid atpB-rbcL intergene has been analyzed within a wide range of plants covering the major dicot lineages. New sequences from 13 plant species were determined and aligned with three already-known sequences. The promoters of the rbcL and the atpB genes were localized and analyzed according to published observations in spinach and tobacco. The evolutionary conservation of two atpB promoters, separated by 113-262 nucleotides, is strong support that both are functionally active, and it also allows a discrimination between the previously reported atpB transcripts. Moreover, the radically distinct sequences of the two atpB promoters suggest that they interact with two distinct initiation complexes. The alignment also confirms the much higher conservation of the leader sequence in the rbcL mRNA than in the atpB mRNA among dicots, presuming a function at the posttranscriptional level.

Molecular phylogeny of families related to Celastrales based on rbcL 5' flanking sequences.

The region between the rbcL and atpB chloroplast genes and the first 53 codons of the rbcL gene have been sequenced for 19 species of angiosperms. Nine of these belong to the four largest families within the order Celastrales sensu Cronquist (i.e., Aquifoliaceae s.l., Icacinaceae, Celastraceae, and Hippocrateaceae). Both phenetic and cladistic approaches were used to test the monophyly of the order and to specify its relationships with Euphorbiaceae, Rhamnaceae, Rosaceae, and Theaceaea. Based upon this molecular analysis, the order Celastrales is polyphyletic and is divided into two major clades. The first group, containing Aquifoliaceae s.l. and Icacina, is related to Camellia (Theaceae). The second, containing Euonymus (Celastraceae), Hippocratea, and Salacia (Hippocrateaceae), is related to Euphorbia (Euphorbiaceae).

A possible explanation for the multiple polyadenylation sites in transcripts coding for a winged-bean leghemoglobin.

Five different copy DNA clones coding for the same leghemoglobin were isolated from a winged-bean (Psophocarpus tetragonolobus L.) nodule library. Although identical in sequence, they each possess a different side of polyadenylation located 93-128 nucleotides downstream of two overlapping AAUAAA putative signal sequences. By analysis of the untranslated 3' ends, a potential mRNA secondary structure can be predicted which could explain the observed polyadenylation heterogeneity. The structure is a size-variable hairpin, creating a net topological distance of 25-27 nucleotides between the canonical signal sequence and the different polyadenylation sites observed. We suggest that this type of variable secondary structure could be one among other causes that determines the apparent flexibility of plant polyadenylation. It could also confer particular properties to the mRNA in relation to stability, translation efficiency and-or nuclear export.

A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor.

Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. Immunocytochemical study showed that the protease inhibitor is exclusively localized in infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata.

Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to "nod-box"-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars.

Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.

Immunocytochemical localisation of lectins in cells of Phaseolus vulgaris L. seeds.

Antibodies against pure E4- and L4-lectins from the seeds of Phaseolus vulgaris L. raised in rabbits were made monospecific by immunoaffinity chromatography on E4- or L4-lectin Sepharose 4B columns. Localisation of lectins in bean seeds was investigated by indirect immunofluorescence and by electron microscopy on sections stained with colloidal gold particles coated with monospecific anti-E4- and anti-L4-IgG. In parenchyma cells from the cotyledons both E- and L-type lectins were found inside the protein bodies. Apparently the matrix of all protein bodies contained both types of lectins. On the other hand in vascular and in axis cells the two types of lectins were localised in the cytoplasm, outside the protein bodies. Thus these findings suggest different roles for the lectins: in cotyledons this may be a specific form of N storage, while in vascular and axis cells lectins may have a more direct metabolic part to play.