Organisation and expression of a wound/ripening-related small multigene family from tomato

cDNA clones derived from a ripe tomato fruit cDNA library were used to investigate changes in the abundance of specific mRNAs in ripening fruit and wounded leaves. mRNAs related to one cDNA clone (pTOM 13) were expressed in both situations. This clone was used to identify homologous sequences in a tomato genomic library. Three groups of related clones that hybridised to the pTOM 13 cDNA insert were identified and subcloned into plasmid vectors. Genomic Southern analysis of tomato DNA using gene-specific DNA fragments isolated from the subcloned DNAs indicated that all pTOM 13 closely related genes had been isolated. RNA dot blot analysis with these DNA fragments as probes indicated differential expression of this small multigene family in leaves and fruit.     

Enzymatic preparation of 1,6-anhydro-muropeptides by immobilized murein hydrolases from Escherichia coli fused to staphylococcal protein A

Summary In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage p _R promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42°C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved. Correspondence to: W. Keck     

Exome sequencing identifies a nonsense mutation in <Emphasis Type="Italic">Fam46a</Emphasis> associated with bone abnormalities in a new mouse model for skeletal dysplasia

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a ^E157*Mhda) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a ^E157*Mhda mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a ^E157*Mhda mice are the first mouse model for a mutation within the Fam46a gene.     

Drought- and frost-induced accumulation of soluble carbohydrates during accelerated senescence in <Emphasis Type="Italic">Quercus pubescens</Emphasis>

Key message

Different environmental conditions affect tree senescence by different patterns of carbohydrate concentrations and have specific impact on the dissection of the photosynthetic apparatus.

Abstract

A proactive cultivation of Mediterranean broadleaf species, including oaks, has been suggested to fill possible temporal and spatial gaps in forestry created by Climate Change in Central Europe in the future. Climate can affect trees in several different ways, e.g., by modulating the course of leaf senescence. Senescence-associated processes, like regulation of carbohydrates and changes in chlorophyll fluorescence under drought stress conditions were studied with leaf tissue of drought-tolerant downy oak (Quercus pubescens). Two months of consistent drought stress in a frost-free greenhouse led to significantly earlier senescence and significant increased amounts of soluble sugars in the leaves of the drought-stressed group in comparison to a well-watered control group. Similar sugar accumulation was observed in trees outdoors, after exposure to frost. In contrast to monocarpic plants the accumulation of free sugars is neither triggering leaf senescence, nor is it a side effect of age-depending changes in Q. pubescens. Instead, sugar accumulation is induced by abiotical factors, like drought and frost. Furthermore, we suggest that the senescence process in the absence of drought stress or frost depends on the source status of the leaf, which, in term, is a function of light (through photosynthesis) and night temperature (through respiration). Contents of the storage metabolite starch decreased during late summer in all three groups. Drought-stressed plants showed a decline of the connectivity of photosystem II antenna, reflected as the L-band in the chlorophyll fluorescence induction curves, and stronger correlations between the declines in the capacity of photosynthetic dark reactions and electron transport-associated chlorophyll fluorescence parameters. We conclude that the disassembly of single parts of the photosynthetic apparatus during leaf senescence is a uniform process, but the onset of this process depends on abiotical environmental factors.

     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Heterozygous expression of X-linked chondrodysplasia punctata

Summary Two females showing partial expression of X-linked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2–Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2–Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2–Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.     

Age-specific patterns in honeydew production and honeydew composition in the aphid Metopeurum fuscoviride: implications for ant-attendance

The intensity of the mutualistic relationship between aphids and ants depends mainly on the composition and amount of honeydew. We used the model system Tanacetum vulgare-Metopeurum fuscoviride to study age-related differences in honeydew production and composition and its effect on the mutualism between M. fuscoviride and the ant Lasius niger. First and second instar larvae of M. fuscoviride produced only half of the amount of honeydew as older larvae or adults. There were, however, no differences between age classes in the total honeydew sugar concentration, which averaged approx. 80 μg sugar/μl honeydew. Honeydew sugar composition also did not differ between age classes, and melezitose was the dominant sugar (59% in all classes). The amino acid concentration, by contrast, increased significantly with aphid age, reaching 22.6 nmol per μl honeydew in adult M. fuscoviride. This increase was mainly caused by asparagine and glutamine, while there were no differences in the concentrations of the five other regularly detected amino acids and cystine, respectively. The intensity of ant-attendance was significantly lower in colonies of first and second instar larvae than in colonies of older age classes. Ant-attendance correlated with the amount of honeydew produced, and not with the total amino acid concentration.     

Effects of Cadmium on Chlorophyll Content,Photochemical Efficiency,and Photosynthetic Intensity of Canna indica Linn.

The effects of cadmium (Cd²⁺) on growth status, chlorophyll (Chl) content, photochemical efficiency, and photosynthetic intensity were studied on Canna indica Linn. Plant specimens that were produced from a constructed wetland and precultivated hydroponically in 20 L of 1/10 Hoagland solution under greenhouse conditions for 1 week were exposed to cadmium in concentrations of 0, 0.4, 0.8, 1.6 and 3.2 mg L^―1 Cd²⁺, respectively. The results show that leaves were injured in the Cd²⁺ solution by the third day of exposure and the injury became more serious with an increase in the applied heavy metal. Under 3.2 mg L^―1 Cd²⁺ treatment, growth retardation, the decrease of chlorophyll content from 0.70 to 0.43 mg g^―1 FW, and a decrease in Chl a/b ratio from 2.0 to 1.2 were observed. Chl a was more sensitive than Chl b to Cd²⁺ stress. The decrease was the same with photochemical efficiency. Photosynthetic intensity decreased by 13.3% from 1.5×10⁴ μmol m^―2s^―1 CO₂ in control to 1.3×10⁴ μmol m^―2s^―1 CO₂ in the treatment of 3.2 mg L^―1. Because Canna species are used in heavy metal phytoremediation, these results show that C. indica can tolerate 0.4 to 0.8 mg L^―1 Cd²⁺. Therefore, it is a potential species for phytoremediation of cadmium with some limitations only at higher concentrations.     

The redox protein construction kit: pre-last universal common ancestor evolution of energy-conserving enzymes

Genome analyses and the resolution of three-dimensional structures have provided evidence in recent years for hitherto unexpected family relationships between redox proteins of very diverse enzymes involved in bioenergetic electron transport. Many of these enzymes appear in fact to be constructed from only a limited set of building blocks. Phylogenetic analysis of selected units from this "redox enzyme construction kit" indicates an origin for several prominent bioenergetic enzymes that is very early, lying before the divergence of Bacteria and Archaea. Possible scenarios for the early evolution of selected complexes are proposed based on the obtained tree topologies.