Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome.

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR-ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST-SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST-SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST-SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST-SSRs each. This unexpectedly high incidence of EST-SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.

Differential gene expression during seed germination in barley (Hordeum vulgare L.).

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process.

Identification of genes specifically expressed in maternal and filial tissues of barley caryopses: a cDNA array analysis.

Developing seeds consist of genetically distinct maternal and filial tissues, whose interactions during development are largely unknown. To better understand the molecular physiology of developing seed tissues in barley, we created a high-density cDNA macroarray bearing 711 cDNA fragments from 691 clones representing at least 620 unique genes mainly derived from a cDNA library constructed with mRNA from the early stages of caryopsis development. This array has been used to compare gene expression patterns in maternal pericarp and filial embryo sac tissues of caryopses sampled 1-7 days after flowering (DAF). The profiles obtained for both tissues revealed that at least 26 genes in pericarp and 12 genes in embryo sac tissues were up-regulated by more than a factor of two during this period. RNAs expressed at high levels in the pericarp mainly encode enzymes involved in carbohydrate and lipid metabolism, but also include mRNA for a transcription factor related to FILAMENTOUS FLOWER (FIL). Genes preferentially expressed in the embryo sac are mainly related to degradation and/or processing of proteins or are involved in the process of starch accumulation, which begins in the seed at this time. Some of the most conspicuously regulated genes were studied in more detail by Northern analysis and in situ hybridization. The mRNA with the highest apparent signal intensity encodes a methionine synthase (MSY). MSY is highly expressed throughout the pericarp and to a lower extent in the transfer cell layer of the endosperm. Of special interest is a gene of unknown function because its high-level expression is restricted to the nucellar projection, the maternal transfer tissue of the caryopsis. This gene, represented by clone HY09L21, may play a central role in transport processes and thus in embryo growth.

Parthenogenetic egg cells of wheat: cellular and molecular studies.

The 'Salmon' system of wheat comprises three isogenic alloplasmic lines with either zygotic (aS) or autonomous, fertilisation-independent (cS kS) embryo development. While the initiation of embryogenesis from the isolated sexual egg cell depends on in vitro fertilisation, the corresponding parthenogenetic egg cell develops into an early embryo without fertilisation. This demonstrates that parthenogenesis is an inherent feature of the isolated egg cell. Based on this observation, we have constructed egg-cell-specific cDNA libraries and report first results of a sequencing project aimed at the isolation of putative egg-cell-specific and parthenogenesis-related genes.

Dynamic interaction of plastocyanin with the cytochrome bf complex.

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.

PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II.

We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane.

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen.

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.

Clp protease complexes and their diversity in chloroplasts.

The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95-00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing "intermediates" and the mature nClpP are found in assembled form.

On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes.

Four distinct integration/translocation routes into/across thylakoid membranes have recently been deduced for nuclear-encoded polypeptides of the photosynthetic membrane. Corresponding information for the plastid-encoded protein complement is lacking. We have investigated this aspect with in-organello assays employing chimeric constructs generated with codoncorrect cassettes for genes of plastid-encoded thylakoid proteins, and appropriate transit peptides from six nuclear genes, representing three targeting classes, as a strategy. The three major plastid-encoded components of the cytochrome b (6)f complex, namely pre-apocytochrome f, (including apocytochrome f, and pre-apocytochrome f lacking the C-terminal transmembrane segment), cytochrome b(6), and subunit IV, which differ in the number of their transmembrane segments, were studied. Import into chloroplasts could be observed in all instances but with relatively low efficiency. Thylakoid integration can occurr post-translationally, but only components with secretory/secretory pathway (SEC)-route-specific epitopes were correctly assembled with the cytochrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b(6) and subunit IV operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably, contribute to this process. The N-termini of cytochrome b(6) and subunit IV appear to reside on the same (lumenal) side of the membrane, consistent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed.

Sodium Dodecyl Sulfate-Stable Proteases in Chloroplasts.

Chloroplast subfractions were monitored for sodium dodecyl sulfate-stable proteases. Nine distinct activities in the molecular mass range from 14 to 66 kD have been detected. Five of the proteases associated with thylakoid membranes belong to the serine and cysteine types of proteases. These activities could be preserved and purified by a two-step electrophoresis procedure.

The plastocyanin binding domain of photosystem I.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.

The 64 kDa polypeptide of spinach may not be the LHCII kinase, but a lumen-located polyphenol oxidase.

Phosphorylation of chlorophyll alb-binding proteins of the of photosystem II light-harvesting assembly controls the energy distribution between the two photosystems as well as the turnover of thylakoid membrane proteins. The LHCII kinase, suggested to be a 64 kDa protein, is light-regulated by a mechanism involving reduction of plastoquinone and the participation of the cytochrome b6lf complex. A cDNA encoding that protein has been isolated from a lambda gt11-based library made from spinach polyadenylated RNA using a two-step strategy involving screening by polyclonal monospecific antisera and plaque hybridization. The protein of 73.1 kDa molecular mass represents a precursor which contains a bipartite transit peptide of 101 amino acid residues (11.0 kDa) that directs the protein into the thylakoid lumen. It can be phosphorylated in vitro, and exhibits significant homology to plant polyphenol oxidases not to kinases. The gene was therefore designated PpoA. Reinvestigation of components in the molecular mass range of 50-70 kDa disclosed five additional proteins which can accompany kinase-active cytochrome b6lf, photosystem II and AMS [1] preparations. Four of them can be phosphorylated in vitro; two with apparent molecular masses of 53 and 66 kDa are capable of phosphorylation and represent new, yet unidentified proteins.

Arabidopsis mutants define downstream branches in the phototransduction pathway.

Light regulates the development of Arabidopsis seedlings in a variety of ways, including inhibition of hypocotyl growth and promotion of leaf development, chloroplast differentiation, and light-responsive gene expression. Mutations that uncouple most or all of these responses from light control have been described, for example, det1, det2, and cop1. To identify regulatory components that define downstream branches in the light-regulated signal transduction pathway, mutants specifically affected in only one light-regulated response were isolated. A screen was designed to isolate mutants that overexpressed the CAB (photosystem II type I chlorophyll a/b-binding proteins) genes in the dark, by use of transgenic line containing a T-DNA construct with two CAB3 promoter-reporter fusions. Eight mutants that showed aberrant expression of both CAB3 promoters were isolated and were designated doc mutants (for dark overepression of CAB). All of the mutants have normal etiolated morphology in the dark. Genetic and phenotypic analyses indicate that most of the mutations are recessive and define at least three loci (doc1, doc2, doc3). Unlike det1 and det2 mutants, which affect the expression of CAB and RBCS (the small subunit of RuBP carboxylase) to approximately the same extent, all three doc mutations are much more specific in derepressing the expression of CAB. The phenotypes of doc mutants suggest that morphological changes can be genetically separated from changes in CAB gene expression. Moreover, the regulation of CAB gene expression can be separated further from the regulation of RBCS gene expression. Epistasis studies suggest that DOC1 and DET3 act downstream from DET1 on two separate branches in the phototransduction pathway. In contrast, DOC2 appears to act on a distinct pathway from DET1. Mutations in doc1, doc2, or doc3 also impair plant growth under short-day conditions.

The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis.

The N-terminal residues preceding the alpha-helix-turn-alpha-helix motif on the Tn10 Tet repressor protein were probed by oligonucleotide-directed deletion mutagenesis for their role in protein activity. All deletion mutants showed decreased repression in vivo, emphasizing the importance of the N terminus for tet operator binding. Only two of the mutants, TetR delta 2-23 and TetR delta 3-8 displayed a reduced intracellular protein level. The remaining deletion mutants showed either reduced binding to tet operator and inducibility by tetracycline or transdominance. We conclude that these deletions do not affect stability and overall protein structure. DNA binding activities of residue-wise increasing deletions, TetR delta 9 through TetR delta 9-13, reveal a pattern consistent with an alpha-helical structure of the affected residues. This conclusion is supported by the helical wheel projection and the hydrophobic moment profile calculated for the protein segment ranging from residues S7-V20. We propose that these residues form an amphipathic alpha-helix which packs closely against the alpha-helix-turn-alpha-helix motif and is essential for Tet repressor activity. The residues preceding this putative alpha-helix contribute to DNA binding, but no direct interactions with base pairs of tet operator were revealed in a loss of contact analysis. Individual mutation of the 4 charged residues to alanine at the N terminus shows that no single residue can account for the reduction in repression observed for the deletion mutants.

A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT.

The tet operators of two naturally evolved tetracycline resistance determinants differ by a G.C to A.T transition at the sixth base pair. This mutation prevents heterologous recognition of these tet operators by their respective two Tet repressor proteins. The amino acid side chains responsible for this sequence-specific distinction of operators were determined. For this purpose in vitro recombinants of the two tetR genes were constructed. Restriction sites were introduced by oligonucleotide-directed mutagenesis in both genes followed by the exchange of different coding segments between them. The encoded chimeric Tet repressor proteins were expressed and their operator recognition specificity was scored in vivo. Exchanging gradually smaller coding segments led finally to a single amino acid exchange in both genes at position 40 of the primary structures. Each Tet repressor containing Thr at this position recognizes the G.C operator while those with Ala recognize the A.T operator regardless of the rest of the sequences. This result demonstrates clearly that the amino acid 40 of Tet repressor contacts and recognizes base pair 6 of tet operator. Sterical interference of the large Thr side chain with the methyl group of A.T and a possible involvement of the hydroxyl in hydrogen bonding to the operator are discussed as the molecular basis of this differentiation between A.T and G.C base pairs.

Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline.

Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.

Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer.

The interaction of Tet repressor protein with the inducer tetracycline was studied by fluorescence measurements, equilibrium dialysis and nitrocellulose filter binding. The repressor-tetracycline complex was formed from two molecules of tetracycline and one Tet repressor dimer. Formation of the complex requires divalent cations, and results in drastic effects upon the fluorescence spectra of both compounds. The fluorescence of Tet repressor was quenched about 70%, while that of tetracycline was increased between three- and eightfold, depending upon pH. In addition, the emission maximum of the protein was shifted from 330 to 340 nm, and the excitation maximum of tetracycline dropped from 380 to 370 nm. The latter shift is accompanied by a similar change in the absorption spectra. An analogous effect was observed upon changing the environment of the drug by the addition of sodium dodecyl sulphate. These results suggest that tetracycline binds to a hydrophobic region of the protein. A new excitation band in the fluorescence spectrum of the complex is observed. This presumably arises from energy transfer from a tryptophan to the drug. The association rate constant for formation of the complex is 3.3(+/- 0.3) X 10(5) M-1 s-1, and the equilibrium association constant is 2.8(+/- 0.5) X 10(9) M-1. These results are discussed with respect to the biological function of the Tet repressor.

Crystallization of and preliminary X-ray diffraction data for TET-repressor and the TET-repressor-tetracycline complex.

The TET-repressor encoded by the transposon Tn10 has been crystallized along with the repressor-tetracycline complex. Both crystals belong to the space group P43212 (or P41212) with cell dimensions a = b = 74.3(1) A, c = 94.2(2) A and a = b = 73.3(1) A, c = 94.6(2) A for the free and complexed repressor, respectively. There is one molecule of molecular weight 23,000 per asymmetric unit, and the biologically active dimer therefore consists of two identically formed subunits which are related by a crystallographic 2-fold axis. This isomorphism of TET-repressor and its tetracycline complex suggests that only minor, subtle changes in structure trigger binding to or release of the operator. The crystals of the native protein permit X-ray data collection to 3.2 A and those of the complexed repressor to 2.8 A.

Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification.

Overproduction of the repressor protein from the Tn10-encoded tetracycline resistance operon is achieved by placement of the respective gene under control of bacteriophage lambda promoter PL in a vector-host system. All cloning steps have to be carried out under repressed conditions to assure survival of the cell. The cI 857 mutation is used to control expression of the tetR gene in large scale fermentation. After induction, the overproducing Escherichia coli strain continues to grow for 2.5 generations before growth terminates. In the expression phase, active TET repressor comprises up to 13% of the total soluble protein. A procedure is described to purify the TET repressor protein to homogeneity on a large scale. Starting from a 10 litre culture, approximately 250 mg of homogeneous, active TET repressor are obtained. The amino acid sequence of the N and C termini are in agreement with the gene start and stop determined from the nucleotide sequence of the Tn10 tetR gene.

TET repressor.tet operator complex formation induces conformational changes in the tet operator DNA.

The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism. For this purpose a 70 bp DNA fragment was prepared which contains both tet operators. About 67% of the base pairs of this DNA are involved in specific interaction with the TET repressor. A rather large change in the CD of the DNA is induced by binding of the TET repressor. The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor. However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA [Culard, F. and Maurizot, J.C. (1981) Nucl. Acids Res. 9, 5157-5184]. Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor.tet operator complexes on polyacrylamide gels under CD conditions, and by a titration experiment of tet operator DNA with TET repressor employing the CD change. The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.