A Pisum sativum glyoxysomal malate dehydrogenase induced by cadmium exposure.

The glyoxysomal malate dehydrogenase (gMDH) catalyses the formation of oxaloacetate from malate during beta-oxidation of fatty acids in the glyoxysome. A partial Pisum sativum L. (cv. Greenfeast) cDNA was first isolated from a suppression subtractive hybridisation cDNA library obtained from heavy metal stressed plants. The full length cDNA was then isolated by rapid amplification of cDNA ends. The translated sequence showed strong similarity to Cucumis sativus and Citrullus lanatus gMDH including a typical glyoxysome-targeting presequence comprising the PTS2 motif and a cleavage site for a cystein-directed protease. Exposure of pea plants to Cd2+ induced expression of the gMDH gene in mature pea leaves indicating that the enzyme is under environmental control in addition to the normal developmental regulation pattern.     

Structure and expression of chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.) isolated by redundant polymerase chain reaction.

Porphobilinogen (PBG) deaminase catalyzes the polymerization of four PBG monopyrrole units into the linear tetrapyrrole hydroxymethylbilane necessary for the formation of chlorophyll and heme in plant cells. Degenerate oligonucleotide primers were designed based on amino acid sequence data (generated by mass spectrometry) for purified PBG deaminase from pea (Pisum sativum L.) chloroplasts. These primers were used in TaqI polymerase-catalyzed polymerase chain reaction (PCR) amplification to produce partial cDNA and nuclear genomic fragments encoding the enzyme. Subsequently, a 1.6-kb cDNA was isolated by screening a cDNA library constructed in lambda gt11 from leaf poly(A)+ RNA with the PCR products. The cDNA encodes an approximately 40-kD polypeptide containing a 46-amino acid NH2-terminal transit peptide and a mature protein of 323 amino acids. The deduced amino acid sequence of the mature pea enzyme is similar to PBG deaminases from other species and contains the conserved arginine and cysteine residues previously implicated in catalysis. Northern blot analysis indicates that the pea gene encoding PBG deaminase is expressed to varying levels in chlorophyll-containing tissues and is subject to light induction.     

Design, expression, and crystallization of recombinant lectin from the garden pea (Pisum sativum)

The propeptide form of the lectin from the garden pea (Pisum sativum agglutinin) has been expressed in Escherichia coli by attaching its cDNA to an inducible promoter. By a number of criteria, including the ability to form dimers, hemagglutination titer, Western blot, and enzyme-linked immunosorbent assay, the resulting propeptide molecule is virtually indistinguishable from the mature proteolytically processed lectin isolated from peas. Preliminary crystallization experiments using the recombinant propeptide lectin yield crystals in space group P2(1)2(1)2(1) with a = 64.8 A, b = 73.8 A, and c = 109.0 A (1 A = 0.1 nm) that diffract to 2.8-A resolution. This unit cell size is quite similar to the unit cell determined for native pea lectin, suggesting that the overall structure of the recombinant prolectin is virtually identical.     

蚕豆豆类胰岛素基因的cDNA克隆及序列分析

为探明豆科植物中豆类胰岛素基因的结构特征与进化关系,在已获得大豆豆类胰岛素基因的基础上,以蚕豆种子胚根mRNA为材料,采用RT-PCR技术,克隆了蚕豆豆类胰岛素基因的cDNA序列,编码的前体多肽包括信号肽、成熟型豆类胰岛素及另一多肽的45个氨基酸残基。DNA序列分析表明,克隆片段与大豆和豌豆的同源性分别为62.5%和58.7%。在氨基酸水平上分别具有44.2%和43.6%的同源性,其中存在着高度保守的半胱氨酸位点,它们在维持豆类胰岛素的空间结构与生理功能方面,可能具有重要的作用。 Abstract:In order to elucidate the relationship between the structural features of leginsulin gene in legume plants and their phylogenetic significance,we have cloned the cDNA sequence of leginsulin gene from radicles of broad bean (Vicia faba) via RT-PCR techniques according to the leginsulin gene sequence we previously obtained from soybean (Glycine max).The cloned cDNA encoded for a precursor protein consisting of the signal peptide,mature leginsulin and an additional 45 amino acids of another polypeptide.A sequence search for homology comparison revealed the cloned leginsulin cDNA fragment shares 62.5% and 58.7% similarity to soybean and pea,respectively.The results also shown that leginsulin cDNA from broad bean presents 44.2% and 43.6% amino acid sequence homology with soybean and pea (Pisum sativum),respectively,and that there exists highly conserved cysteine sites among the leginsulin cDNAs,which may play a crucial role in maintaining the three-dimensional structure and the physiological functions of leginsulin.     

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning,localization, and putative function

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.     

The rug3 locus of pea encodes plastidial phosphoglucomutase

Two cDNA clones were isolated from pea (Pisum sativum L.) and their deduced amino acid sequences shown to have significant homology to phosphoglucomutases from eukaryotic and prokaryotic sources. The longer cDNA contained a putative transit-peptide-encoding sequence, supporting the hypothesis that the isolated clones represent the cytosolic and plastidial isoforms of phosphoglucomutase in pea. Plastid protein import assays confirmed that the putative plastidial isoform was targeted to the plastid stroma where it was proteolytically processed. Expression, co-segregation, linkage, and molecular analyses have confirmed that the rug3 locus of pea encodes plastidial phosphoglucomutase. Mutations at this locus result in a near-starchless phenotype of the plant.     

cDNA cloning, primary structure and gene expression for H-protein, a component of the glycine-cleavage system (glycine decarboxylase) of pea (Pisum sativum) leaf mitochondria.

We have isolated and characterized cDNA clones encoding the H-protein of the glycine-cleavage system of pea (Pisum sativum) leaf mitochondria. The deduced primary structure revealed that the 131-amino-acid polypeptide is cytoplasmically synthesized with a 34-amino-acid mitochondrial targeting peptide. The lipoate-binding site was assigned to be lysine-63, as deduced from a sequence comparison with several lipoate-bearing proteins. The expression of the gene encoding H-protein was shown to occur specifically in the leaf tissue, with light exerting an additional effect by increasing the mRNA levels severalfold. Two polyadenylation sites were found in the mRNA, and a single-copy gene encoding the H-protein was detected in pea genome.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.     

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.     

Isolation of a cDNA clone for pea (Pisum sativum) seed lipoxygenase.

A lipoxygenase cDNA clone, pCD45, was identified in a Pisum sativum L. (pea) seed mRNA cDNA library by hybrid-release/translation followed by immunoprecipitation with antiserum raised against lipoxygenase from Glycine max L. (soya bean). pCD45 hybrid-selected an mRNA encoding the larger of the two polypeptides of Mr approximately 95 000 that were immunoprecipitated from cell-free translation products of pea seed poly(A)-containing RNA by the G. max anti-lipoxygenase. 'Northern'-blot analysis showed the mRNA that hybridized to pCD45 to be approximately 3000 nucleotides in length. Three to five copies of the lipoxygenase gene corresponding to pCD45 were estimated to be present per haploid Pisum genome; hybridization of the cDNA insert from pCD45 to G. max DNA was also detected.     

Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica)

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al.. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.     

Chloroplast and cytoplasmic enzymes: isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases.

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.     

Dual intracellular localization and targeting of aminoimidazole ribonucleotide synthetase in cowpea

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 ATP-dependent 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.     

Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis

Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.     

Expression of delta1- and delta3-cyclin genes in pea (pisum sativum L.) root meristem during clinorotation

Using in situ hybridization the delta1- and delta3-cyclin gene expression has been shown in pea (Pisum sativum L.) root meristem cells under slow horizontal clinorotation and in the stationary conditions. The clinorotation effect on expression of these genes during pea root germination was detected. The delay of degradation of cyclin subunits is the cause of G1-phase prolongation under clinorotation leading to delay of pea seed germination. This may explain the increase of cell cycle duration.     

Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter.

DNA sequences encoding ribulose 1,5-bisphosphate carboxylase small subunit precursor from Pisum sativum L. have been transcribed from plasmids containing the SP6 promoter, and translated in a wheat germ cell-free system. The small subunit precursor polypeptide, its N-terminal leader sequence (transit peptide) and the mature small subunit have each been synthesized independently from three different plasmid constructs. The precursor polypeptide is imported into isolated pea chloroplasts and processed to the mature small subunit by a stromal proteinase. The mature polypeptide is neither imported, nor subject to proteolysis by stromal extracts. The transit peptide alone is very rapidly degraded by a stromal proteinase activity which can be inhibited by EDTA or 1,10-phenanthroline. The use of these gene constructs helps to establish the crucial role of the transit peptide in protein import into the chloroplast.