Immunoelectrophoretic analysis of seed proteins from Pisum sativum L.

Summary Soluble proteins of pea seed were investigated by quantitative immunological methods. Vicilin, legumin, pea seed lectin (PEA), 26 albumins and a globulin (B₁) were detected and observed during seed development, germination and under different extraction and fractionation procedures. Vicilin and legumin were found to be immunologically distinctly different. Legumin was found to be comprised of two similar proteins, Legumin species I and II. Vicilin, but no legumin, was detected in the embryonic axis.Three albumins, B₁ and PEA were found to be synthesized after the onset of legumin synthesis.Among the pea lines investigated, one line exhibited distinct differences with respect to the albumins and PEA.Some observations indicate that PEA might interact with other seed proteins of pea.     

Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein

A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast. These proteins belong to the ras superfamily of proteins involved in different basic cellular processes. The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle. The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively. The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence. However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation.Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots.     

Gibberellin induces endopeptidase activity in detached cotyledons of Pisum sativum

Endopeptidase activity in cotyledons of 5-day seedlings of Pisum sativum increased rapidly during germination. However, the increase of the activity in detached cotyledons was depressed. We examined whether a growth regulator can be substituted for the embryonic axis on the development of endopeptidase activity. As monitored by an assay with azoalbumin, the development of endopeptidase activity from crude extracts of detached cotyledons appeared to be slightly accelerated by incubation with 10^–5 M GA₃. However, the pattern after gelatin-polyacrylamide gel suggested that the activity induced in detached cotyledons during a 5-d incubation at 10^–7 M GA₃ was the same as that in attached ones during germination for 5 days and an even greater increase in activity was obtained with 10^–5 M GA₃. These results suggest that GA₃ from the embryonic axis induces endopeptidase activity in attached cotyledons at the first stage of germination.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - GA gibberellic acid     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.     

Molecular cloning,sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.     

A novel ras-related rgp1 gene encoding a GTP-binding protein has reduced expression in 5-azacytidine-induced dwarf rice.

Summary Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Shizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling growth, rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development.     

Proximity of an ARS consensus sequence to a replication origin of pea (Pisum sativum)

The replication origin (ori-r9) of the 9.0 kb rDNA repeats of pea (Pisum sativum, cv. Alaska) was cloned and found to reside in a 1.5 kb fragment of the non-transcribed spacer region located between the 25 S and 18 S genes. Labeled rDNA rich in replication forks, from cells positioned at the G₁/S phase boundary, was used to map ori-r9 by hybridization procedures. Ori-r9 is in a 210-base fragment that is 1.6 kb from the 5 end of the 18 S gene and about 1.5 kb from the 3 end of the 25 S gene. The same procedures, using labeled synthetic ARS consensus sequence as a probe, showed than an ARS consensus sequence is located 3 to ori-r9 in a 710-base fragment. An ARS consensus sequence is, therefore, adjacent to ori-r9 but not coincidental with it.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Transformation of peas (Pisum sativum L.) using immature cotyledons

A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 mg/l phosphinothricin. The nptII and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the Buster (=Basta) leaf-paint test and the phosphinothricin acetyl transferase enzyme assay.Abbreviations BA 6-benzylaminopurine     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems

Summary The chromosomal status of calli and plantlets regenerated fromPisum sativum shoot apical meristems was studied. Chromosome mosaicism (aneusomaty) occurs during callus induction and proliferation, mostly owing to nuclear fragmentation prior to mitosis in the first days of culture. Plantlets regenerated from calli are diploid or aneusomatic, but a selective advantage of diploid cells (diplontic selection) takes place with plantlet growth. The results are discussed in relation to the possibility of inducing chromosomal and/or genetic variability by using meristematic tissues as expiants.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H+-ATPase

Summary Evidence suggests that certain ras-related small molecular weight GTP-binding proteins (smg-proteins) are involved in intracellular membrane trafficking and vesicle fusion. We have previously shown that intravesicular acidification due to a vacuolar-type H⁺-ATPase, which is Cl^– dependent and highly sensitive to the specific inhibitor bafilomycin, enhances GTP-induced fusion of pancreatic microsomal vesicles (Hampe, W., Zimmermann, P., Schulz, I. 1990. FEBS Lett. 271:62–66). This process may involve function of smg-proteins. The present study shows that MgATP (2 mm), but neither MgATPS nor ATP in the absence of Mg²⁺, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [-³²P]GTP binding. The affinity of smg-proteins for [-³²P]GTP was not altered by MgATP. Bafilomycin B₁ (10^–8 m), the protonophore CCCP (10^–5 m), and replacement of Cl^– in the incubation buffer by CH₃COO^– or NO ₃ ^– resulted in an almost complete inhibition of the MgATP-dependent association of the 19- and 21-kDa smg-proteins to the vesicle membranes. Furthermore, the MgATP effect on both smg-proteins was found to be due to the intravesicular pH and not to the H⁺ gradient over the vesicle membrane. We conclude that association of a 19-kDa (immunologically identified as the ADP-ribosylation factor, arf) and a yet unidentified 21-kDa GTP-binding protein to vesicle membranes is regulated by the intravesicular pH established by a vacuolar-type H⁺-ATPase.The arf-antibodies were kindly supplied by Dr. R.A. Kahn. We thank Prof. Dr. D. Gallwitz, Dr. R. Jahn, and Dr. E.G. Lapetina for kindly providing the ypt 1-, rab 3-, and rap 1-antibodies, respectively. ADP-ribosyltransferase C₃ from Clostridium botulinum was kindly supplied by Prof. Dr. K. Aktories. This work was supported by the Jung-Stiftung für Wissenschaft und Forschung. S.Z. was supported by a grant of the Deutsche Forschungsgemeinschaft (Ze 237/3-1).     

An altered constitutive peptide in sym 5 mutants of Pisum sativum L.

Mutational analysis of Pisum sativum L. was used to search for constitutive proteins that might function in nodule formation. The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation. Comparison of two-dimensional polyacrylamide gels of in vitro-translated root RNA showed a consistent difference in the migrational pattern of one peptide. In the nodulating parental cultivar Sparkle, a 66 kDa peptide had a pI of 5.9. In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8. This 66 kDa peptide is found in lateral root, tap root, and shoot. Its expression was independent of rhizobial inoculation, root temperature, or light.     

Organization of the genes encoding chalcone synthase in Pisum sativum

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.     

Inheritance and mapping of isoenzymes in pea (Pisum sativum L.)

Summary Three isoenzyme systems (amylase, esterase and glutamate oxaloacetate transaminase) were examined in seeds of pea (Pisum sativum L.) and shown to give clear variation in their band patterns on gel electrophoresis between different lines. The inheritance of these isoenzyme systems, and the location of their genes on the pea genome was investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase was found to be on chromosome 2, linked to the loci k and wb (wb ... 9 ... k ... 25 ... Amy). The gene for esterase was found to be linked with the gene Br (chromosome 4) but the exact location is uncertain because of the lack of the morphological markers involved in the cross. The gene for glutamate oxaloacetate transaminase was found to be on chromosome 1 and linked with the loci a and d (a... 24... Got... 41 ... d).     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

Biologically Active Oligosaccharides from Pectins of Pisum sativum L. Seedlings Affecting Root Generation

Two physiologically active oligosaccharide fractions were isolated from pectin of Pisum sativum L. cell wall after its partial acid hydrolysis. These fractions displayed stimulating and inhibiting effects on root formation in thin-layer explants. The subsequent separation of these fractions by gel permeation and anion-exchange chromatography resulted in fractions with effective concentrations two orders of magnitude lower than the concentrations of the initial fractions. The resulting oligosaccharides displayed their effect on the earliest stage of the rhizogenesis associated with formation of root primordias. The rhizogenesis-inhibiting fraction suppressed cell division by 30-50%. The stimulating fraction mainly contained fragments of xyloglucan and galactan, and the inhibiting fraction contained fragments of xyloglucan, galactan, and arabinan. The polymerization degrees of the stimulating and of the inhibiting oligosaccharides were 10-11 and 5-6, respectively.