Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Nucleolar transformation in plants grown on clinostats

Summary Cells of carrot calli (Daucus carota L.) grown on clinostats (simulated weightlessness) exhibit increases in nucleolar number and volume. In clinostat-grown whole barley plants (Hordeum vulgare L. cv. Steptoe), nucleoli in 70% of root meristem and root cortical cells in the 1 mm root apex exhibit multiple nodulations after one day of growth. The nucleolar nodules (1.1 m mean diameter) are densely and finely fibrous, distinctly different from the nucleolus in which the content is so compact that the granular component is masked. Control nucleoli (from vertically rotated and stationary seedlings) rarely exhibit nodule-like protrusions, are not compact, and contain a well defined granular component. Proteins that are heat soluble, characteristic of many stress responses, rapidly increase in barley grown on clinostats. Barley growth on clinostats is slowly and steadily inhibited. There is no difference between vertically rotated and stationary controls for any of the parameters measured, indicating that clinostat motion per se does not affect significantly barley development. The evidence taken together suggests that barley plants germinated and grown on clinostats are stressed, the effects of which are expressed sequentially by alteration of nucleolar morphology, increased production of heat-soluble proteins, and decreased plant growth. Similar stress-related changes may be expected to occur in plants subjected to weightlessness during space flight. It is therefore of interest that nucleoli in wheat roots (Triticum aestivum L. cv. Broom) obtained from the space flight IML-1 mission show irregularity that is not observed in any of the ground controls for the flight experiment.Abbreviations Act D actinomycin D - C clinostat rotation - EM electron microscopy - LM light microscopy - R vertical rotation - rDNA ribosomal DNA - S stationary     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Water-soluble proteins of mature barley endosperm: genetic control,polymorphism, and linkage with β-amylase and spring/winter habit

Summary Water-soluble proteins (WSP-2 and WSP-3) and -amylase (-AMY-1) were extracted from mature endosperms of 44 spring and 39 winter barley genotypes. The protein and enzyme isoforms were separated in isoelectric focusing gels with a pH gradient of 4–6.5. The Wsp-3 and -Amy-1 loci were located to chromosomes 4H using the wheat/barley chromosome addition lines. Segregation analysis of F₂ and doubled haploid populations showed Wsp-2 and -Amy-1 to be tightly linked, with a map distance of 11 cMorgans. Isoforms of WSP-2 possessed similar pIs to that of WSP-3 and overlapping bands were observed in the gels. These bands segregated independently in F₂ and doubled haploid populations, implying two unlinked genes. All three loci were found to be polymorphic: two alleles were detected at the Wsp-2 locus, three at Wsp-3 and two at -Amy-1. The frequency of alleles at all three loci was found to be different in winter and spring genotypes. Spring genotypes possessed a wider range of phenotypes than winter genotypes. Spring and winter genotypes could be distinguished on the basis of WSP-3 and - AMY-1 phenotypes. The linkage between Wsp-3 and -Amy-1 loci and genes controlling spring/winter habit on chromosome 4H is discussed. It is concluded that Wsp-3 and -Amy-1 can be used as genetic markers for spring/winter habit in barley genetic research and breeding.     

Seven members of the (1→3)-β-glucanase gene family in barley (Hordeum vulgare) are clustered on the long arm of chromosome 3 (3HL)

Members of the (13)--glucan glucanohydrolase (EC 3.2.1.39) gene family have been mapped on the barley genome using three doubled haploid populations and seven wheat-barley addition lines. Specific probes or polymerase chain reaction (PCR) primers were generated for the seven barley (13)--glucanase genes for which cDNA or genomic clones are currently available. The seven genes are all located on the long arm of chromosome 3 (3HL), and genes encoding isoenzymes GI, GII, GIII, GIV, GV and GVII (ABG2) are clustered in a region less than 20 cM in length. The region is flanked by the RFLP marker MWG2099 on the proximal side and the Barley Yellow Mosaic Virus (BYMV) resistance gene ym4 at the distal end. The gene encoding isoenzyme GVI lies approximately 50 cM outside this cluster, towards the centromere. With the exception of the gene encoding isoenzyme GIV, all of the (13)--glucanase genes are represented by single copies on the barley genome. The probe for the isoenzyme GIV gene hybridized with four DNA bands during Southern blot analysis, only one of which could be incorporated into the consensus linkage map.     

A low-temperature-responsive translation elongation factor 1α from barley (Hordeum vulgare L.)

A cDNA clone (pBLT63) encoding a protein synthesis elongation factor 1 (EF-1) was isolated from a low-temperature winter barley shoot meristem library by differential screening. The nucleotide sequence of the coding region of the low-temperature-induced barley gene shows very high homology with two EF-1 plant genes from tomato and Arabidopsis. The barley genome contains an EF-1 gene family situated on the short arm of chromosome 2 and the long arm of chromosome 5. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Z23130.     

Polymorphisms in the α-amy1 gene of wild and cultivated barley revealed by the polymerase chain reaction

-Amylases are the key enzymes involved in the hydrolysis of starch in plants. The polymerase chain reaction (PCR) was used to detect polymorphisms in the length of amplified sequences between the annealing sites of two primers derived from published -amy1 gene sequences in barley. These two primers (Bsw1 and Bsw7), flanking the promoter region and the first exon, amplified two PCR fragments in barley. One of the amplified products, with the expected length of 820 bp, appeared together with another shorter PCR band of around 750 bp. This 750-bp fragment seems to be derived from an -amylase gene not reported previously. Both of the PCR products could be amplified from the two-rowed barley varieties tested, including cv Himalaya from which the sequence information was obtained. Five of the six-rowed barley varieties also have the two PCR fragments whereas another two have only the long fragment. These two fragments seem to be unique to barley, neither of them could be amplified from other cereals; for example, wheat, rye or sorghum. These two -amylase fragments were mapped to the long arm of 6H, the location of the -amy1 genes, using wheat-barley addition lines. Amplification of genomic DNA from wild barley accessions with primers Bsw1 and Bsw7 indicated that both of the fragments could be present, or the long and short fragments could be present alone. The results also demonstrated that the genes specifying these two fragments could be independent from each other in barley. The conserved banding pattern of these two fragments in the two-rowed barley varieties implies that artificial selection from these genes may have played an important role in the evolution of cultivated barley from wild barley.     

Improvement of β-amylase thermostability in transgenic barley seeds and transgene stability in progeny

The genetic improvement of enzymes important in the brewing process is one of the main goals of barley biotechnology. For the improvement of -amylase thermostability in barley seeds, we have already constructed a mutant thermostable -amylase gene, using site-directed mutagenesis and random mutagenesis to achieve the substitution of seven amino acids of the original barley -amylase. This sevenfold-mutant barley -amylase showed a thermostability increased by 11.6 °C compared to the original enzyme. In the present article, a thermostable -amylase gene under the control of the barley -amylase promoter was introduced to barley protoplasts, and fertile plants were generated from 9 independent transgenic lines. Subsequent analyses indicated that the thermostable -amylase gene was expressed and -amylase activity derived from both native and modified genes was detected in the seeds of 6 transgenic lines. The transgene was stably transmitted to progeny, and thermostable -amylase was synthesized in T₄ seeds, demonstrating that our strategy is applicable for the improvement of seed quality for industrial utilization.     

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3--glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and pathogenesis-related proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.Abbreviations BMT S-adenosyl-L-methionine: bergaptol O-methyltransferase (EC 2.1.1.-) - 4CL 4-coumarate: CoA ligase (EC 6.1.1.12) - CMT S-adenosyl-L-methionine: caffeate O-methyltransferase (EC 2.1.1.-) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - PR pathogenesis-related - XMT S-adenosyl-L-methionine: xanthotoxin O-methyltransferase (EC 2.1.1.-)     

Positive φ-angles in proteins by nuclear magnetic resonance spectroscopy

Summary Non-glycine residues with positive -angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) ofBacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant and integration of the nuclear Overhauser H^N-H cross peak, positive -angles could be determined reliably to 60°±30°, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive -angles can also occur in non-glycine residues and in the four proteins, positive -angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured coupling constants and the intensity of the intraresidue H^N-H NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wüthrich, K. (1984)J. Mol. Biol.,180, 741–751; Ludvigsen, S., Andersen, K.V. and Poulsen, F.M. (1991)J. Mol. Biol.,217, 731–736).