Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis in Amaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

Induction of secondary dormancy in Amaranthus caudatus seeds

Nondormant A. caudatus seeds germinated in the darkat temperatures between 20 and 35° but not at 45 °C.Incubation at this temperature for at least 10 h inhibited seedgermination over the temperature range 20 to 35 °C,temperatures previously suitable for germination. Thus incubation at 45°C induced secondary dormancy. Mechanical or chemicalscarification or exposure to pure oxygen caused complete or almost completegermination of dormant seeds although more slowly in comparison to nondormantseeds. Secondary dormant scarified seeds required a lower concentration of ABAthan nondormant seeds to inhibit germination. The high temperature, whichinduced dormancy, 45 °C, caused the seed coat to be partiallyresponsible for secondary dormancy. Involvement of ABA (synthesis orsensitivity) in the induction and/or maintenance of this dormancy should beconsidered.     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Molecular Cloning of a cDNA Encoding Ribosome Inactivating Protein from Amaranthus viridis and Its Expression in E. coli

In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis     

Implication of Ethylene in the Release of Secondary Dormancy in Amaranthus caudatus L. Seeds by Gibberellins or Cytokinin

Ethephon (Eth), gibberellin A₃, A_{4 + 7} (GA₃, GA_{4 + 7}), and 6-benzyladenine (BA) removed secondary dormancy of Amaranthus caudatus seeds. The GAs and BA potentiated the effect of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene biosynthesis precursor, in terms of the rate or final percent of germination. Aminoethoxyvinylglycine (AVG), an ACC synthase activity inhibitor, was observed to simultaneously inhibit the release from dormancy effected by GA₃ or BA as well as the ethylene production stimulated by these regulators. Breaking of secondary dormancy by GA₃, GA_{4 + 7} or BA was prevented by 2,5-norbornadiene (NBD), an inhibitor of ethylene binding. Ethylene completely or markedly reversed the inhibitory effect of NBD. We thus conclude that the removal of secondary dormancy in Amaranthus caudatus seeds by gibberellin or benzyladenine involves ethylene biosynthesis and action.     

An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds

A 30-residue antimicrobial peptide Ar-AMP was isolated from the seeds of amaranth Amaranthus retroflexus L. essentially by a single step procedure using reversed-phase HPLC, and its in vitro biological activities were studied. The complete amino acid sequence of Ar-AMP was determined by Edman degradation in combination with mass spectrometric methods. In addition, the cDNA encoding Ar-AMP was obtained and sequenced. The cDNA encodes a precursor protein consisting of the N-terminal putative signal sequence of 25 amino acids, a mature peptide of 30 amino acids and a 34-residue long C-terminal region cleaved during post-translational processing. According to sequence similarity the Ar-AMP belongs to the hevein-like family of antimicrobial peptides with six cysteine residues. In spite of the fact that seeds were collected in 1967 and lost their germination capacity, Ar-AMP retained its biological activities. It effectively inhibited the growth of different fungi tested: Fusarium culmorium (Smith) Sacc., Helminthosporium sativum Pammel., King et Bakke, Alternaria consortiale Fr., and Botrytis cinerea Pers., caused morphological changes in Rhizoctonia solani Kühn at micromolar concentrations and protected barley seedlings from H. sativum infection.     

NO donors mimic and NO inhibitors inhibit cytokinin action in betalaineaccumulation in Amaranthus caudatus

A classical biotest for cytokinin is based on the accumulation of betalainesin Amaranthus species. We have shown that inhibitorsof nitric oxide synthase from animals inhibit this response and that chemicaldonors of nitric oxide (NO) stimulate betalaine biosynthesis in Amaranthuscaudatus. NO could be an intermediate in cytokinin signalling.     

Typification of seabeach amaranth, Amaranthus pumilus (Amaranthaceae)

Amaranthus pumilus is known from coastal Massachusetts to South Carolina and from ballast in Philadelphia, Pennsylvania. It is currently listed by the United States Fish and Wildlife Service as a threatened species.Amaranthus pumilus was originally described by Rafinesque in 1808 from southern New Jersey, but he did not cite specimens. Here, the nameA. pumilus is lectotypified using the only known original element, a Rafinesque specimen at the herbarium of The Academy of Natural Sciences, Philadelphia (PH).     

Cloning and characterization of a cDNA encoding a Rab1-like small GTP-binding protein from Petunia hybrida

The cloning of small GTP-binding proteins from Petunia hybrida was performed using a PCR-based strategy. Degenerate primers were designed from the DTAGQE and FMETSA consensus sequences. Three different cDNAs were amplified. The deduced polypeptide sequences PhPCRGP1 and PhPCRGP2 were homologous to RB11_HUMAN and PhPCRGP3 to RAB1A_HUMAN. Using PhPCRGP3 as a probe, 8 identical clones were selected from a Petunia leaf cDNA library. They all encode the same 22.5 kDa polypeptide, PhRAB1, able to bind GTP in vitro and 72% identical to RAB1A_HUMAN. Hybridizable mRNAs encoding PhRAB1 accumulated preferentially in opened flowers.     

银杏种仁中抗菌蛋白的纯化及性质

银杏果仁提取上清液经硫酸铵沉淀、CM—52离子交换柱层析和Sephadex G-50凝胶过滤层析后分离纯化出一种抗菌蛋白。SDS—PAGE分析结果表明,此蛋白分子量为13000,对热稳定;氨基酸组分分析表明,该蛋白含18种不同氨基酸,尤富含丙氨酸(Ala)和精氨酸(His)等,缺乏半胱氨酸(Cys);纯化的蛋白对黄瓜镰刀孢菌(Fusarium oxysporum)、瓜类炭疽菌(Colletotrichum orbiculare)、小麦全蚀病菌(Gaeumannomyces graminis)等真菌有很强的抑制作用,而且对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia Coli)等细菌也有一定的抑制作用。     

Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein

A Triticum durum cDNA library prepared from developing endosperm (22 days after flowering (DAF)) was screened using synthetic oligonucleotide probes covering part of the CM3 and CM16 N-terminal protein sequences. A full-length cDNA clone (pTd78) encoding the CM16 protein (chloroform/methanol-soluble protein) was isolated and characterized. To our knowledge this is the first characterization of a clone coding for a wheat CM protein. The CM16 protein is synthesized as a preprotein with a signal peptide of 24 residues, the molecular weight of the mature protein being 13 438 Da. As other members of the cereal trypsin/-amylase inhibitor family, the CM16 protein contains 10 cysteine residues, their position being well conserved. In developing endosperm the highest level of CM16 mRNA was detected at mid-maturation.     

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.