Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO₂ assimilation measured in individual leaves was substantially diminished only under conditions of high CO₂ and low O₂. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod. Received: 4 January 2000 / Accepted: 21 March 2000     

Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) somatic embryogenesis from isolated scutellum: Days post anthesis,days of spike storage,and sucrose concentration affect efficiency

Summary To improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l⁻¹ casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.     

Expression and secretion of barley α-amylase and A.niger glucoamylase in Saccharomyces cerevisiae

cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol. Project supported by the Guangdong Natural Science Foundation.     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

Relationships of endogenous plant hormones to accumulation of grain protein and starch in winter wheat under different post-anthesis soil water statusses

Accumulation of protein and starch in grain is a key process determining grain yield and quality in wheat. Under drought or waterlogging, endogenous plant hormone levels will change and may have an impact on the yield and quality of wheat. In a greenhouse experiment, four winter wheat (Triticum aestivum L.) varieties differing in grain protein content, Heimai 76, Wanmai 38, Yangmai 10 and Yangmai 9, were subjected to drought (SRWC = 4550%, DR), waterlogging (WL) and moderate water supply (SRWC = 7580%, CK), beginning from 4 days post-anthesis (DPA) to maturity. On the 10 (grain enlargement stage) and 20 (grain filling stage) DPA, endogenous abscisic acid (ABA), gibberellins (GA₁₊₃), indole-3-acetic acid (IAA) and zeatin riboside (ZR) were determined in sink and source organs of wheat plants by enzyme linked immunosorbent assay (ELISA). The patterns of hormonal changes were similar in four varieties. The ABA levels were much higher under DR and WL than under CK. Compared with CK, GA₁₊₃ levels in whole-plant under DR and WL changed a little at 10 DPA, but markedly decreased under DR and WL at 20 DPA. Changes of endogenous IAA level under DR and WL exhibited a complicated pattern, depending on organs and growth stages. Particularly at the 20 DPA, the mean levels of IAA in roots, leaves and grains decreased significantly under DR and WL. In comparison with CK, ZR levels in all organs significantly decreased under DR and WL at both stages. The correlation analyses between yields and contents of starch and protein in grains and levels and ratios of four hormones in source and sink organs indicated that the changes in yield and content of grain starch and protein under DR and WL were associated with the reduced IAA, ZR and GA₁₊₃ levels and elevated ABA level in plants, especially in grains. It was proposed that the changed levels of endogenous hormones under post-anthesis DR and WL might indirectly affect protein and starch accumulation in grains by influencing the regulatory enzymes and processes.     

Conservation and change in structural and 5′ flanking sequences of esterase 6 in siblingDrosophila species

Esterase 6 (Est-6/EST6) is the major β-carboxylesterase inD. melanogaster and its siblingsD. simulans andD. mauritiana. It is expressed in several tissues but its major site of expression is the sperm ejaculatory duct of the adult male. Although EST6 activity affects reproductive fitness, there are high levels of electrophoretic and activity polymorphism, at least withinD. melanogaster andD. simulans. Here we present the nucleotide sequences of anEst-6 allele and its flanking regions from each ofD. simulans andD. mauritiana and compare them with the publishedD. melanogaster sequences. As might be expected, replacement sites are significantly less divergent than exon silent sites in all comparisons, suggesting that selection is acting to maintain EST6 structure and function among the three species. Nevertheless, the ratio of the levels of replacement to silent site divergence is still much higher forEst-6 than for seven of ten other genes (including both isozyme-coding loci) for which comparable data have been published for these species. This is consistent with the high levels of EST6 electrophoretic polymorphism withinD. melanogaster andD. simulans and implies that selective constraints against amino acid change are relatively weak for EST6. By contrast, comparisons involving promotor sequences show that the level of divergence in the first 350bp 5′ of the gene is significantly lower than those for four of the six other loci for which comparable data have been published for these species. In particular, there are two perfectly conserved stretches (−1 to −158bp and −219 to −334bp) each over 100bp long included in this 350bp region. Thus the data suggest a relatively low level of selective constraint on the amino acid sequence of EST6 but a relatively high level of constraint on sequences affecting aspects of its expression.     

Developmental mutants ofStreptomyces coeruleorubidus,a producer of anthracyclines: Isolation and preliminary characterization

Abstbact The wild strainStreptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 andbld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variantbld-1 after treatment with mutagens (UV light, γ radiation, nitrous acid) and after natural selection Formation of the aerial mycelium was fully suppressed in thebld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; typesspo-2, spo-3, spo-4 andspo-5 differed in size, shape and surface structure of spores, the typewhi formed asporogenous aerial hyphae.     

Molecular analysis of an HLA-DP mutant cell line selected for its resistance to killing by HLA-DPw2-specific T-cell clones

A collection of HLA-DP mutants was generated, using ICR 191 as the mutagenic agent and resistance to lysis mediated by HLA-DPw2 allospecific cytotoxic T lymphocytes (CTLs) as the selection criterion. These mutants were derived from the HLA haploid lymphoblastoid cell line 45.1. Loss of HLA-DPw2 surface expression accounted for the lack of HLA-DPw2 CTL recognition in all the mutants. However, one of them, 45.EM19, binds to DPw2-specific monoclonal antibodies (mAb) after cell permeabilization. HLA-DPA1 and DPB1 mRNA expression studies permitted the classification of the mutants in four categories: 1) DPA1-negative, DPB1-positive; 2) DPA1-positive, DPB1-negative; 3) DPA1- and DPB1-negative, and 4) DPA1- and DPB1-positive mutants. Mutant 45.EM19 is included in the last group. The cloning and sequencing of the full-length DPA1 (DPA1*0103) and DPB1 (DPB1*02012) cDNAs from this mutant showed no changes in the DPA1 sequence compared to the wild-type sequence. However, a frame-shift mutation in the DPB1 gene exon coding for the transmembrane region was detected. The insertion of a guanine nucleotide provokes an extension of the open reading frame, increasing the length of the C-terminal domain and changing the hydropathicity pattern of the transmembrane domain. This change should be responsible for the phenotype of the 45.EM19 mutant. Correspondence to: Dr. M. Sánchez-Pérez.     

Starch-bound 2S proteins and kernel texture in einkorn, <Emphasis Type="Italic">Triticum</Emphasis> <Emphasis Type="Italic">monococcum</Emphasis> ssp <Emphasis Type="Italic">monococcum</Emphasis>

The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE. All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B), as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured common wheat, “monococcum” accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules, respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele Eti-A ^m 1a and “valine-type” ETI encoded by allele Eti-A ^m 1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low as −2.05 ± 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in “monococcum” wheat, as determined by visual assessments in counting chambers, was estimated at 764 mm²/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification of factors that cause the extra-soft texture of einkorn kernels.