Gunther Neuhaus

Cyclic GMP and Calcium Mediate Phytochrome Phototransduction

We have previously used single-cell assays in a phytochrome-deficient tomato mutant to demonstrate that phytochrome signaling involves heterotrimeric G proteins, calcium, and calmodulin. While G protein activation could stimulate full chloroplast development and anthocyanin pigment biosynthesis, calcium and calmodulin could not induce anthocyanins and were only able to stimulate the development of immature chloroplasts lacking cytochrome b6f and photosystem I core components. We now report that cyclic GMP is able to trigger the production of anthocyanins, and that a combination of cyclic GMP with calcium can induce the development of fully mature chloroplasts containing all the photosynthetic machinery. Furthermore, using reporter genes for these different pathways (cab-gus, chs-gus, and fnr-gus) we demonstrate that cGMP and calcium act primarily by modulating gene expression.

Calcium/calmodulin-dependent and -independent phytochrome signal transduction pathways

Phytochrome is a well-characterized plant photoreceptor, able to modulate many morphological, physiological, and biochemical events through as yet undefined mechanisms. By developing single-cell assays to visualize phytochrome responses, we have studied the effects of microinjecting putative signaling intermediates into phytochrome-deficient tomato cells. We demonstrate that phytochrome phototransduction initially involves the activation of one or more G proteins that are coupled to at least two different pathways; one pathway requires calcium and activated calmodulin and can stimulate expression of a photoregulated cab-GUS reporter gene together with the synthesis and assembly of some, but not all, of the photosynthetic complexes. The other pathway, controlling anthocyanin biosynthesis, does not require calcium. Furthermore, our results reveal that phytochrome signaling is cell autonomous and is not likely to require any light-activated steps downstream of the G protein.

HLM1, an Essential Signaling Component in the Hypersensitive Response, Is a Member of the Cyclic Nucleotide–Gated Channel Ion Channel Family

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide–gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K+ and Na+ and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.

Phytochrome‐regulated repression of gene expression requires calcium and cGMP

The plant photoreceptor phytochrome A utilizes three signal transduction pathways, dependent upon calcium and/or cGMP, to activate genes in the light. In this report, we have studied the phytochrome A regulation of a gene that is down‐regulated by light, asparagine synthetase (AS1). We show that AS1 is expressed in the dark and repressed in the light. Repression of AS1 in the light is likely controlled by the same calcium/cGMP‐dependent pathway that is used to activate other light responses. The use of the same signal transduction pathway for both activating and repressing different responses provides an interesting mechanism for phytochrome action. Using complementary loss‐ and gain‐of‐function experiments we have identified a 17 bp cis‐element within the AS1 promoter that is both necessary and sufficient for this regulation. This sequence is likely to be the target for a highly conserved phytochrome‐generated repressor whose activity is regulated by both calcium and cGMP.

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G‐proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light‐inducible wheat Cab‐1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis‐related (PR) protein genes encoding PR‐1 and the class II isoforms of PR‐2 and PR‐3. In contrast, the class I isoforms of PR‐2 and PR‐3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP‐gamma‐S induced the expression of a PR1‐GUS reporter gene but not that of a GLB‐GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR‐2. Microinjection and grafting experiments strongly suggest that CTX‐sensitive G‐proteins are important in inducing the expression of a subset of PR genes and that these G‐proteins act locally rather than systemically upstream of SA induction.

A novel phytyltransferase from Synechocystis sp. PCC 6803 involved in tocopherol biosynthesis

The deduced polypeptide sequence of open reading frame slr1736 reveals homology to chlorophyll synthase and 1,4‐dihydroxy‐2‐naphthoic acid phytyltransferase in Synechocystis sp. strain PCC 6803. In tocopherol and plastoquinone biosynthesis, a condensation reaction mechanistically similar to that of these two enzymes is performed. To analyze the function of this novel prenyltransferase, a deletion mutant of slr1736 was generated by homologous recombination. The mutant showed a markedly decreased tocopherol content, while plastoquinone levels remained unchanged. Since the aromatic precursor homogentisic acid accumulated in the mutant, the function of the enzyme was proven to be a novel tocopherol phytyltransferase.

Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana.

The recently identified cyclic nucleotide‐gated ion channels (AtCNGCs) from Arabidopsis thaliana have the ability to bind calmodulin. Using two different methods, we mapped the binding site of AtCNGC1 to the last predicted α helix of the cyclic nucleotide binding domain. This is in contrast to CNGCs from animals, where the calmodulin binding site is located in the N‐terminus, implying that different mechanisms for CNGC modulation have evolved in animals and plants. Furthermore, we demonstrate that AtCNGC1 and AtCNGC2 have different calmodulin binding affinities and we provide evidence for target specificities among calmodulin isoforms.

Plasma Membrane H + -ATPase Is Involved in Auxin-Mediated Cell Elongation during Wheat Embryo Development

Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H+-ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two–three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H+-ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H+-ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H+-ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.

Interleukin-6 Stimulates LDL Receptor Gene Expression via Activation of Sterol-Responsive and Sp1 Binding Elements

Inflammatory or malignant diseases are associated with elevated levels of cytokines and abnormal low density lipoprotein (LDL) cholesterol metabolism. In the acute-phase response to myocardial injury or other trauma or surgery, total and LDL cholesterol levels are markedly decreased. We investigated the effects of the proinflammatory cytokine interleukin (IL)-6 on LDL receptor (LDL-R) function and gene expression in HepG2 cells. IL-6 dose-dependently increased the binding, internalization, and degradation of (125)I-LDL. IL-6-stimulated HepG2 cells revealed increased steady-state levels of LDL-R mRNA. In HepG2 cells transiently transfected with reporter gene constructs harboring the sequence of the LDL-R promoter extending from nucleotide -1563 (or from nucleotide -234) through -58 relative to the translation start site, IL-6 dose-dependently increased promoter activity. In the presence of LDL, a similar relative stimulatory effect of IL-6 was observed. Studies using a reporter plasmid with a functionally disrupted sterol-responsive element (SRE)-1 revealed a reduced stimulatory response to IL-6. In gel-shift assays, nuclear extracts of IL-6-treated HepG2 cells showed an induced binding of SRE binding protein (SREBP)-1a and SRE binding protein(SREBP)-2 to the SRE-1 that was independent of the cellular sterol content and an induced binding of Sp1 and Sp3 to repeat 3 of the LDL-R promoter. Our data indicate that IL-6 induces stimulation of the LDL-R gene, resulting in enhanced gene transcription and LDL-R activity. This effect is sterol independent and involves, on the molecular level, activation of nuclear factors binding to SRE-1 and the Sp1 binding site in repeat 2 and repeat 3 of the LDL-R promoter, respectively.

Induction of zygotic polyembryos in wheat: influence of auxin polar transport

The effects of two auxin polar transport inhibitors, N-1-naphthylphthalamic acid (NPA) and 3,3[prime],4[prime],5,7-pentahydroxyflavone (quercetin), on attaining bilateral symmetry from radial symmetry during early wheat embryogenesis were investigated by using an in vitro culture system. Although NPA and quercetin belong to two different classes of auxin transport inhibitors, the phytotropins and the flavonoids, respectively, they induced the same specific abnormal phenotypes during embryo development. These abnormal embryos differentiated multiple meristems (i.e., multiple shoot and root meristems) and multiple organs (i.e., multiple coleoptiles and scutella). Multiple shoot apical meristem phenotypes were characterized by partly multiplied embryonic axes and supernumerary scutella. The differentiation of multiple primary roots in addition to multiple shoot meristems and multiple scutella led to the formation of polyembryos. The occurrence of multiple shoot meristem phenotypes depended on the concentration of the inhibitor and the developmental stage of the isolated embryo. Embryos treated with NPA or quercetin developed multiple radicle phenotypes less frequently than they developed multiple shoot meristem phenotypes. Our observations suggest that the root meristem differentiates later than the shoot meristem. Our data support the hypothesis that polar transport of auxin has a determining influence on the differentiation of the embryonic axis and the scutellum.