Oxidative Heck desymmetrisation of 2,2-disubstituted cyclopentene-1,3-diones.

Oxidative Heck couplings have been successfully developed for 2,2-disubstituted cyclopentene-1,3-diones. The direct coupling onto the 2,2-disubstituted cyclopentene-1,3-dione core provides a novel expedient way of enantioselectively desymmetrising all-carbon quaternary centres.

Activation tagging in Arabidopsis.

Activation tagging using T-DNA vectors that contain multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene has been applied to Arabidopsis plants. New activation-tagging vectors that confer resistance to the antibiotic kanamycin or the herbicide glufosinate have been used to generate several tens of thousands of transformed plants. From these, over 30 dominant mutants with various phenotypes have been isolated. Analysis of a subset of mutants has shown that overexpressed genes are almost always found immediately adjacent to the inserted CaMV 35S enhancers, at distances ranging from 380 bp to 3.6 kb. In at least one case, the CaMV 35S enhancers led primarily to an enhancement of the endogenous expression pattern rather than to constitutive ectopic expression, suggesting that the CaMV 35S enhancers used here act differently than the complete CaMV 35S promoter. This has important implications for the spectrum of genes that will be discovered by this method.

Characterization of a gene encoding a DNA-binding protein that interacts in vitro with vascular specific cis elements of the phenylalanine ammonia-lyase promoter.

A study of the expression of a bean phenylalanine ammonia-lyase (PAL) promoter/beta-glucuronidase gene fusion in transgenic tobacco has shown that the PAL2 promoter has a modular organization. Expression of the PAL2 promoter in the vascular system involves positive and negative regulatory cis elements. Among these elements is an AC-rich motif implicated in xylem expression and a suppressing cis element for phloem expression. Using radiolabelled complementary oligonucleotides bearing the AC-rich motif, a cDNA clone encoding a DNA-binding protein has been isolated from a tobacco lambda gt11 expression library. This factor, named AC-rich binding factor (ACBF), showed binding specificity to the AC-rich region. The specificity of ACBF for the AC-rich region was also shown using a gel retardation assay with an ACBF recombinant protein extract. The deduced amino acid sequence from ACBF contains a long repeat of glutamine residues as found in well characterized transcription factors. Interestingly, ACBF shared sequence similarity to conserved amino acid motifs found in RNA-binding proteins. Genomic gel blot analysis indicated the presence of a small gene family of sequences related to ACBF within the tobacco nuclear genome. Analysis of tobacco mRNA using the ACBF cDNA as probe showed that while ACBF mRNA was present in all tissues examined, the highest transcript accumulation occurred in stem tissues. The functional characteristics of the AC-rich sequence were examined in transgenic tobacco. A heptamer of the AC-rich sequence, in front of a minimal 35S promoter from cauliflower mosaic virus (-46 to +4), conferred specific expression in xylem.

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression.

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of beta-D-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)7CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.

Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses--a review.

In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.

Stress responses in alfalfa (Medicago sativa L.). XX. Transcriptional activation of phenlpropanoid pathway genes in elicitor-induced cell suspension cultures.

Nuclear transcript run-on analysis was used to investigate++ the relative transcription rates of genes encoding enzymes of isoflavonoid phytoalexin biosynthesis and related pathways in elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures. Genes encoding L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone reductase (CHR) were most rapidly activated, with increases in transcription measurable within 10-20 min after elicitation. Cinnamic acid 4-hydroxylase (C4H), chalcone isomerase (CHI), isoflavone reductase (IFR) and caffeic acid 3-0-methyltransferase (COMT) genes were also rapidly activated, but at a slower initial rate. Transcription of chalcone 2'-O-methyltransferase (CHOMT), and 1,3-beta-D-glucanase genes was less rapid, with lag periods of 60 and 30 min post-elicitation, respectively. Treatment of cells with a PAL inhibitor L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) resulted in increased transcription of PAL, CHS and CHR, but reduced transcription of CHOMT, indicating a role for phenylpropanoid products as both negative and positive regulators of gene expression within the phenylpropanoid pathway.

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter.

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5' to 3') in the 5' region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter-beta-glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position -173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the -326 to -173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position -173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.

Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis.

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid synthesis. The role of PAL in pathway regulation was investigated by measurement of product accumulation as a function of enzyme activity in a collection of near-isogenic transgenic tobacco plants exhibiting a range of PAL levels from wild type to 0.2% of wild type. In leaf tissue, PAL level is the dominant factor regulating accumulation of the major product chlorogenic acid and overall flux into the pathway. In stems, PAL at wild-type levels contributes, together with downstream steps, in the regulation of lignin deposition and becomes the dominant, rate-determining step at levels 3- to 4-fold below wild type. The metabolic impact of elevated PAL levels was investigated in transgenic leaf callus that overexpressed PAL. Accumulation of the flavonoid rutin, the major product in wild-type callus, was not increased, but several other products accumulated to similarly high levels. These data indicate that PAL is a key step in the regulation of overall flux into the pathway and, hence, accumulation of major phenylpropanoid products, with the regulatory architecture of the pathway poised so that downstream steps control partitioning into different branch pathways.

Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products.

It has been proposed that natural products synthesized by plants contribute to their resistance to pests and pathogens. We show here that transgenic tobacco plants with suppressed levels of the phenylpropanoid biosynthetic enzyme phenylalanine ammonia-lyase (L-phenylalanine ammonia-lyase, EC 4.3.1.5) and correspondingly low levels of chlorogenic acid, the major soluble leaf phenylpropanoid product, exhibit more rapid and extensive lesion development than wild-type plants after infection by the virulent fungal pathogen Cercospora nicotianae. These observations provide direct evidence that phenylpropanoid products contribute to disease limitation. No induction of transcripts encoding phenylalanine ammonia-lyase or the lignin branch pathway enzyme caffeic acid O-methyltransferase was observed during the infection and there was no perturbation in the pattern of soluble phenylpropanoids. Hence, increased disease susceptibility does not involve inhibition of a pathogen-induced response but likely reflects inhibition of the developmental accumulation of chlorogenic acid. Demonstration of the contribution of such preformed protectants to plant health identifies attractive targets for manipulation by breeding or gene transfer to reduce the quantitative impact of disease.

atpk1, a novel ribosomal protein kinase gene from Arabidopsis. II. Functional and biochemical analysis of the encoded protein.

The Arabidopsis Atpk1 protein expressed in insect cells and plant cells exhibited multiple sizes consisting mainly of two doublets: p70 (68 and 70 kDa) and p85 (82 and 85 kDa). Extraction of p85 from cells required the presence of SDS, suggesting that p85 is associated with less soluble subcellular components. p70 was extracted by nonionic detergent without SDS, indicating that this form is cytoplasmic. p70 expressed in either Arabidopsis or insect cells underwent serine-specific autophosphorylation, indicating that Atpk1 is a protein-serine kinase. A point mutation (lysine 163 to arginine) in the ATP-binding site of the catalytic domain substantially diminished activity when expressed in insect cells. A 14-kDa protein (p14) was co-immunoprecipitated with p70 from insect cells expressing wild-type Atpk1 and was phosphorylated in immune complex kinase assays with Atpk1, suggesting it is a homolog of a natural substrate of Atpk1. Two plant ribosomal proteins (14 and 16 kDa) can be phosphorylated by the Atpk1 protein kinase, and we propose that Atpk1 is a novel ribosomal protein kinase. A 60-kDa form of Atpk1 derived from the insect cell-expressed p70 was more highly phosphorylated than p70 in in vitro kinase assays, suggesting a negative regulatory domain can be removed by proteolysis.

atpk1, a novel ribosomal protein kinase gene from Arabidopsis. I. Isolation, characterization, and expression.

Two protein kinase genes (atpk1 and atpk2) were isolated from Arabidopsis thaliana genomic DNA with a probe generated by polymerase chain reaction (PCR) using oligonucleotide primers encoding conserved eukaryotic protein kinase sequences. atpk1 and atpk2 are organized in a head-to-tail tandem array on chromosome 3 and have about 80% nucleotide sequence identity. atpk1 encodes a hydrophilic polypeptide of 465 amino acids, M(r) = 52,554. The centrally located catalytic domain contains all the conserved residues characteristic of eukaryotic protein kinases, with greatest similarity to the catalytic domains of 70-kDa ribosomal S6 protein kinase, protein kinase C, and protein kinase A. The C-terminal 75 residues also show homology to protein kinase C and S6 protein kinase. In contrast, the N-terminal 130 residues have no homology to any known protein, and thus may represent a new class of protein kinase regulatory domain. Other motifs found in the Atpk1 protein include two putative autophosphorylation sites, a pseudosubstrate site, two acidic domains, a lysine-rich domain, and two putative PEST sequences, which may contribute to the regulation of protein kinase activity. RNA-blot hybridization showed that atpk1 encoded a 1.8-kb mRNA. Analysis of atpk1 promoter/beta-glucuronidase reporter gene fusions in transgenic plants showed that atpk1 was expressed in all tissues and at all developmental stages, with the strongest expression observed in metabolically active tissues, suggesting that atpk1 is involved in the control of plant growth and development. The first intron of atpk1 functions as an enhancer in atpk1 expression.

Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible.

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5' end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogen Magnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.

PAD1 encodes phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in Saccharomyces cerevisiae.

The yeast enzyme phenylacrylic acid decarboxylase (PAD) confers resistance to phenylacrylic acids. Cinnamic acid (CA)-sensitive mutants lacking PAD activity were isolated and the PAD1 gene was cloned by phenotypic complementation. The nucleotide sequence of the smallest complementing fragment was determined. The predicted 242-amino-acid PAD polypeptide is 48.6% identical to the product of dedF of Escherichia coli. PAD activity and CA resistance, but not steady-state PAD1 mRNA levels, are influenced by mitochondrial genotype. PAD1 is a single-copy gene in the yeast genome and not essential for viability. The PAD1 locus was physically mapped to a position approx. 140 kb from the left end of chromosome IV.

In vitro heart valve testing: steady versus pulsatile flow.

The design of artificial heart valves has traditionally been based on the development of a prototype device which was then subjected to extensive laboratory testing in order to confirm its suitability for clinical use. In the past the in vitro assessment of a valve's performance was based principally on the measurement of parameters such as pressure difference, regurgitation and, more recently, energy losses. Such measurements can be defined as being at the 'macro' level and rarely show any clinically significant differences amongst currently available prostheses. The analytical approach to flow through heart valves has previously been hampered by difficulties experienced in solving the relevant equations of flow particularly in the case of pulsatile conditions. Computational techniques are now available which enable appropriate solutions to be obtained for these problems and consequently provide an opportunity for detailed examination of the 'micro' level of flow disturbances exhibited by the different valves. This present preliminary study is designed to illustrate the use of such an analytical approach to the flow through prosthetic valves. A single topic has been selected for this purpose which is the comparative value of steady versus pulsatile flow testing. A bileaflet valve was chosen for the analysis and a mathematical model of this valve in the aortic position of the Sheffield Pulse Duplicator was created. The theoretical analysis was carried out using a commercially available Computational Fluid Dynamics package, namely, FIDAP, on a SUN MICROSYSTEMS 10-30 workstation.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbial recognition and activation of plant defense systems.

Molecules released or generated during microbial entry (elicitors) are recognized by components of plant cells, ultimately resulting in the induction of a battery of plant defense responses. The molecular mechanisms underlying these signaling systems, as well as the plant defense responses they control, are becoming increasingly well characterized.

Hormone-inducible expression and metal affinity chromatography of recombinant proteins in Saccharomyces cerevisiae.

An economical method to express recombinant protein in yeast (Saccharomyces cerevisiae) was developed. We combined two principles: Ni(2+)-agarose-based affinity purification of fusion proteins and expression from a cassette regulated by steroid hormones. After induction by desoxycorticosterone, > 90% pure protein was obtained after a single round of metal affinity chromatography. Gentle lysis conditions allowed the preservation of enzymatic activity. Average yields of 10 mg protein per liter of culture were obtained at a fraction of the cost of other eukaryotic expression systems.

Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes.

The H-box (CCTACC(N)7CT(N)4A), which occurs three times within the -154 to -42 region of the bean chalcone synthase chs15 promoter, is important for developmental regulation of chs15, and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP-1 and KAP-2, which recognize conserved features in the H-box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP-1 is a 97 kDa polypeptide, whereas KAP-2 comprises two polypeptides of 76 and 56 kDa. KAP-1 and KAP-2 also differ in the sensitivity of their DNA-bound forms to trypsin. Dephosphorylation of KAP-1 or KAP-2 affects the mobility of the protein/H-box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP-1 or KAP-2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.

Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system.

CHS15 is one of a family of bean genes encoding chalcone synthase, which catalyzes the first reaction in a branch pathway of phenylpropanoid biosynthesis for the production of flavonoid pigments and UV protectants and isoflavonoid-derived phytoalexins. The functional architecture of the CHS15 promoter was dissected by a novel homologous plant in vitro transcription initiation system in which whole-cell and nuclear extracts from suspension-cultured soybean cells direct accurate and efficient RNA polymerase II-mediated transcription from an immobilized promoter template. Authentic transcription from the CHS15 promoter template was also observed with whole-cell extracts from suspension-cultured cells of bean, tobacco, and the monocot rice, and the soybean whole-cell extract transcribed several other immobilized promoter templates. Hence, this procedure may be of general use in the study of plant gene regulation mechanisms in vitro. Assay of the effects of depletion of the soybean whole-cell extract by preincubation with small regions of the CHS15 promoter or defined cis elements showed that trans factors that bind to G-box (CACGTG, -74 to -69) and H-box (CCTACC, -61 to -56 and -121 to -126) cis elements, respectively, make major contributions to the transcription of the CHS15 promoter in vitro. Both cis element/trans factor interactions in combination are required for maximal activity. Delineation of these functional cis element/trans factor interactions in vitro provides the basis for study of the mechanisms underlying developmental expression of CHS15 in pigmented petal cells established by G-box and H-box combinatorial interactions, and for characterization of the terminal steps of the signal pathway for stress induction of the phytoalexin defense response.