The Endosperm and the Embryo of Arabidopsis thaliana are Independently Transformed through Infiltration by Agrobacterium tumefaciens

Several experiments had indicated that in planta transformation of Arabidopsis thaliana by Agrobacterium involves the female germ line. In order to identify the precise stage at which transformation occurs we have monitored expression of a gusA reporter gene in the two products of the double fertilization of infiltrated plants. The plantlets and the remaining endosperm of seeds were separately tested after germination. It appeared that in the majority of cases only the plantlet or the endosperm were transformed. Based on transformation with two vectors borne by two different Agrobacterium strains, the minority of co-transformed plantlets and endosperm can be explained by simultaneous but independent transformation events. These results indicate that mature female gametes could be the targets of T-DNA.     

A study of integrative transformation in Schizosaccharomyces pombe

Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.     

The Drosophila melanogaster homologue of the hsp60 gene is encoded by the essential locus l(1)10Ac and is differentially expressed during fly development

 The hsp60 (heat-shock protein 60) gene family of molecular chaperones has been a subject of study in numerous systems due to its important role in the correct folding of non-native proteins in development as well as after heat-shock treatment. Here we present the characterization of the first Drosophila hsp60 homologue. Drosophila HSP60 is most closely related (72% identity across the entire protein sequence) to the mouse mitochondrial HSP60. Western blot experiments indicate that Drosophila HSP60 is enriched in the mitochondrial fraction. The distribution of HSP60 protein is dynamic during fly embryogenesis, suggesting that various cell types might have different HSP60 requirements. The molecular analysis of a P-element-induced mutation that affects the l(1)10Ac locus shows that the transposon is inserted in a 3-kb intron present in the hsp60 gene. By genetic rescue experiments we prove that Drosophila HSP60 is encoded by the essential locus l(1)10Ac opening the possibility for detailed genetic analysis of HSP60 functions in the fly. Received: 24 March 1997 / Accepted: 16 June 1997     

Heat shock protein HSP60 can alleviate the phenotype of mitochondrial RNA-deficient temperature-sensitive mna2 pet mutants

mna2, which belongs to the class I temperature-sensitive pet mutants that lose mitochondrial (mt)RNA at restrictive temperature, was shown by complementation and sequence determination to correspond to the gene coding for HSP60. Both mna2-1 and mna2-2, the two available alleles of mna2, have conservative single amino acid substitutions in the HSP60 gene. Valine substitutes for an alanine (position 47) in mna2-1, and an isoleucine substitutes for a valine (position 77) in mna2-2. These substitutions result in defects in respiration and in steady-state mtRNA accumulation. Wild-type hsp60 alleviates the mtRNA phenotype completely, while partially relieving the respiratory deficiency.     

Mutations that are synthetically lethal with a<Emphasis Type="Italic"> gas1Δ</Emphasis> allele cause defects in the cell wall of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The GAS1 -related genes of fungi encode GPI-anchored proteins with -1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1 allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1 cells confirmed the important role of -1,6-glucan in cells that are defective in the processing of -1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1 defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1 mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2 mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1 cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.Communicated by C. P. Hollenberg     

Heat shock pre-treatment enhances the response of Arabidopsis thaliana leaves and Cucurbita pepo cotyledons to benzyladenine

The effects of heat shock (HS) pre-treatment on the response tobenzyladenine were studied in two plant model systems (1) retardation ofsenescence of Arabidopsis thaliana L. Heyhn rosette leavesand (2) induction of greening of detached Cucurbita pepoL.cotyledons. N⁶-benzyladenine (BA) retarded senescence of rosetteleaves of Arabidopsis thaliana (L) Heyhn and briefpre-treatment with HS (3 at 37)essentially enhanced this cytokinin effect. BA stimulated cotyledon greening inCucurbita pepo L due to the activation of chlorophyllsynthesis. Brief cotyledon pre-heating at moderate temperatures (3 at 33–35) also enhanced thiscytokinin effect.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

Rearrangement,amplification, and assortment of mitochondrial DNA molecules in cultured cells of Brassica campestris

Summary We compared Brassica campestris mitochondrial and chloroplast DNAs from whole plants and from a 2-year-old cell culture. No differences were observed in the chloroplast DNAs (cpDNAs), whereas the culture mitochondrial DNA (mtDNA) was extensively altered. Hybridization analysis revealed that the alterations are due entirely to rearrangement. At least two inversions and one large duplication are found in the culture mtDNA. The duplication element is shown to have the usual properties of a plant mtDNA high frequency recombination repeat. The culture mtDNA exists as a complex heterogeneous population of rearranged and unrearranged molecules. Some of the culture-associated rearranged molecules are present in low levels in native plant tissue and appear to have sorted out and amplified in the culture. Other mtDNA rearrangements may have occurred de novo. In addition to alterations of the main mitochondrial genome, an 11.3 kb linear mtDNA plasmid present in whole plants is absent from the culture. Contrary to findings in cultured cells of other plants, small circular mtDNA molecules were not detected in the B. campestris cell culture.     

Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins.

We have created yeast strains in which the mitochondrial chaperonin, hsp60, can be either physically depleted or functionally inactivated. Cells completely depleted of hsp60 stop growing but retain for awhile the capacity to reaccumulate hsp60. While this newly made hsp60 is targeted to and processed correctly within the mitochondrion, assembly of a functional hsp60 complex does not occur. Rather, the hsp60 monomers are localized in different-size soluble complexes containing another mitochondrial chaperone, the mitochondrial form of hsp70. A number of other mitochondrial matrix-targeted proteins synthesized in the absence of functional hsp60 are imported into mitochondria but often show some buildup of precursor forms and, unlike hsp60, accumulate as insoluble aggregates. By contrast, several mitochondrial proteins normally targeted to the intermembrane space show normal processing in the complete absence of a functional hsp60 complex. Similar and complementary results were obtained when we examined the metabolism of matrix- and intermembrane space-localized proteins in cells expressing three different temperature-sensitive alleles of HSP60. In all cases, matrix-targeted proteins synthesized at nonpermissive (i.e., hsp60-inactivating) temperatures were correctly targeted to and processed within mitochondria but accumulated predominantly or totally as insoluble aggregates. The metabolism of two intermembrane space proteins, cytochrome b2 and cytochrome c1, was unaffected at the nonpermissive temperature, as judged by the correct processing and complete solubility of newly synthesized forms of both proteins. These findings are discussed with regard to current models of intermembrane targeting.     

Transient expression of β-glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun

Successful transient expression of -glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm²) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm² of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm² of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.     

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene -glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and -amylase inhibitors.     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).     

Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis

The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains.To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble -phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplast cpn10 proteins in two cases restored -phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn 10 showed significantly reduced complementation efficiencies.     

Interactions of Bovine Brain Tubulin with Pyridostigmine Bromide and N,N′-Diethyl-m-Toluamide

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent K _d of about 60 M. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.     

Structures of the carbohydrate units of polysialoglycoproteins isolated from the eggs of four species of salmonid fishes

Fish egg polysialoglycoprotein (PSGP) is a novel type of 200 kDa-glycoprotein containing more than 50% sialic acid by weight and about 90O-glycosidically-linked sialoglycan units per molecule. Of about 100 different molecular species assumed to be present in a sialoglycan mixture obtained by alkaline borohydride treatment ofSalvelinus leucomaenis pluvius PSGP, 23 mono- to tetrasialylglycans were isolated by anion-exchange chromatography and preparative column chromatography on porous silica, and their structures were determined. Core asialo-oligosaccharides were obtained from PSGP of four species of salmonid fishes by exhaustive enzymatic desialylation of sialoglycan mixtures and the structures of purified compounds were determined. Two complete types of asialopentasaccharide core structures, Fuc1-3GalNAc1-3Gal1-4 Gal1-3GalNAcOL, and GalNAc1-4GalNAc1-3Gal1-4Gal1-3GalNAcOL, and all of the possible biosynthetic precursors of these pentasaccharide cores were found in every PSGP examined. All types of oligosaccharide chains, both complete and incomplete, were found to occur in highly sialylated forms in PSGP.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - GalNAcOL N-acetylgalactosaminitol - PSGP polysialoglycoprotein - SRO sialidase-resistant oligosaccharide     

Function of the maize mitochondrial chaperonin hsp60: specific association between hsp60 and newly synthesized F1-ATPase alpha subunits.

Mitochondria contain a protein, hsp60, that is induced by heat shock and has been shown to function as a chaperonin in the assembly of mitochondrial enzyme complexes composed of proteins encoded by nuclear genes and imported from the cytosol. To determine whether products of mitochondrial genes are also assembled through an interaction with hsp60, we looked for association between hsp60 and proteins synthesized by isolated mitochondria. We have determined by electrophoretic, centrifugal, and immunological assays that at least two of those proteins become physically associated with hsp60. In mitochondrial matrix extracts, this association could be disrupted by the addition of Mg-ATP. One of the proteins that formed a stable association with hsp60 was the alpha subunit of the multicomponent complex F1-ATPase. We have not identified the other protein. These results indicate that hsp60 can function in the folding and assembly of mitochondrial proteins encoded by both mitochondrial and nuclear genes.     

Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter--glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.     

Mitochondrial DNA diversity in the genera of Triticum and Aegilops revealed by southern blot hybridization

Summary Southern blot hybridization of total DNA to defined mitochondrial DNA sequences provides a sensitive assay for mtDNA variation in the genera of Triticum and Aegilops. A clear distinction between cytoplasms of tetraploid species sharing the AG haploid genome is reported for the first time. The Sitopsis section of the genus Aegilops showed the most extensive intra- and inter-specific variation, whereas no variation could be detected among the cytoplasms of polyploid Triticum species (wheats) sharing the AB haploid genome. Extensive cytoplasmic intraspecific diversity was revealed in Ae. speltoides.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.