Calcium/Calmodulin-Dependent and Independent Phytochrome Signal Transduction Pathways

Transcription

1 Calcium/Calmodulin-Dependent and Independent Phytochrome Signal Transduction Pathways Gunther Neuhausl, Chris Bowler2, Rainer Kern2, and Nam-Hai Chua2 1Institut fur Pflanzenwissenschaften, ETH-Zentrum, Universitatstrasse 2, CH-8092 Zurich, Switzerland 2Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York We have developed single cell assays to visualize phytochrome responses by studying the effects of microinjecting putative signalling intermediates into phytochrome-deficient tomato cells. We demonstrate that phytochrome phototransduction initially involves G protein activation and that, subsequently, there are two different pathways - one pathway requires calcium and activated calmodulin, and can stimulate expression of a photoregulated 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. The existence of these general signalling pathways has also been shown in a photomixotrophic soybean cell culture by studying the effects of pharmacological antagonists and agonists on light responsive gene expression. These complementary studies may suggest the involvement of more than one G protein. Furthermore, our results reveal that phytochrome signalling is cell autonomous and is not likely to require any light-activated steps downstream of the G protein

2 STRATEGIES FOR CONTROL OF FUNGAL DISEASES WITH TRANSGENIC PLANTS. Leo S. Melchers, Peter J.M. van den Elzen, Rob A. Schiloeroort. and Erik Jonaediik. MOGEN Int. N.V.. Einsteinwea 97, 2333 CB Leiden, The Netherlands. A promising novel strategy for crop protection is the engineering of durable resistance in plants by the introduction of genes coding for antifungal proteins. Pathogenesis related proteins, isolated from TMV inoculated leaves of tobacco (Samsun NN) were tested for antifungal activity. In vitro studies demonstrated that specifically the tobacco class I (vacuolar) chitinases (Chi-I) and class I B- 1,3-glucanases (Glu-I) are potent inhibitors of Fusarium species and act synergistically, as illustrated in Figure 1. In contrast, the tobacco class II chitinases (Chi-II) and class II B-1,3-glucanases (Glu-II), which are localized extracellularly, display in vitro no antifungal activity, neither alone nor in combination with other proteins (Figure 1). We have studied the possibility to increase fungal resistance of plants by simultaneously introducing genes coding for tobacco chitinases and B-1,3- glucanases.in vivo studies demonstrated a substantially increased resistance against Fusarium oxysporum f.sp. lycopersici in transgenic tomato plants expressing both the tobacco class I chitinase and class I B-1,3-glucanase gene, as shown in Figure 2. Transgenic tomato plants expressing either of these genes alone, showed no increased resistance to F.oxysporum. Our in vivo results provide the first experimental support to the hypothesis that synergistic activity of chitinases resistance in planta. and 3-1,3-glucanases contribute to enhanced fungal Figure 1. Antifungal activity of mixtures of purified tobacco chitinases and &1,3-glucanases on Fusarium solani in vitro. A mounts of protein in ug per well are indicated. Figure 2. Resistance of iransgenic tomato plants expressing class I chitinase and class I f1-1,3-glucanase genes to Fusarium oxysporum f.sp. lycopeisici. Moneymaker control (left) and transgenic line (right), 40 days post-infection

3 EXPRESSION AND GENETIC STABILITY IN TRANSGENIC PLANTS. Lambert A.M. Hensgens and Rob A. Schilaeroort. Leiden University AL Leiden, The Netherlands. Phenotypic expression of the introduced trait and stable transmisssion of this trait to the progeny is the ultimate goal of plant transformation. Our reporter gene fusions studies in tobacco and rice have shown that the phenotypic expression is both dependent on the promoter and initiation of translation sequences. The steady state level and turnover of the mrna and protein, however, also play crucial roles in determining the phenotype. These levels of expresssion control are all differentially regulated in different cell types and are dependent on the growth and differentiation state of the cells. Regeneration of rice was hampered by transformation but not by in vitro growth. Transgenic plants often displayed high frequency of aberrrant pollen and very low seed set or complete sterility. Control and "escape" plants displayed normal pollen development and seedset. The disturbed seed and pollen development was strictly correlated with the transgenic state. Southern analysis showed the presence of large multimeric inserts in the Ro and frequent loss of the inserts in their R, upon selfing. This suggests that transgenesis leads to chromosome aberrations. A literature review of monocot transgenesis and the transfer of the inserted genes to the progeny (selfing and crosses) suggests that chromosomal deletions are linked to the transgenes. These deletions appear to be independent of crop, transformation method and DNA used. We are currently developing systems to circumvent this deletion event to increase plant (cell) vigour and efficiency of transgenesis. Left pannel: Phenotypic GUS expression (staining with X-Gluc) of rice callus transformed with a rice gene GOS2-gusA fusion. the level of mrna of the rice GOS2 gene was shown earlier to be similar for all tissues studied. Despite this the ultimate phenotypic expression of the fusion is highly celltype specific. Flight pannel: Histochemical GUS assay on pollen in an anther of a transgenic rice plant showing an abnormal GUS+ phenotype segregation and pollen development

4 MOLECULAR MECHANISMS OF PROLIFERATION AND DIFFERENTIATION IN PLANT CELL CULTURES. Atsushi Komamine,* Tohoku University, Sendai 980, Japan Plant cell cultures are useful tools for investigation on molecular mechanisms of proliferation and differentiation of higher plant cells, because it is possible to establish high frequency and synchronous systems by using cell cultures. We established synchronous cell division cultures of Catharanthus roseus cells which were induced by double phosphate starvation and auxin starvation methods. Using these systems, we isolated phase-specific cdnas and investigated the expression of the these genes, PCNA and cyclins in the cell cycle of C. roseus. One of isolated cdnas, designated cyc 07, was expressed specifically during the S phase, coupling with DNA synthesis. cyc 07 protein was localized in nuclei of proliferating cells. The homologous genes to cyc 07 were cloned in yeast. Results of gene disruption and tetrad analysis indicated that these genes encode an essential protein for cell division in yeast. cyc 07 could replace the function of yeast genes, suggesting that cyc 07 may play a similar role in C. roseus. We also established a high frequency and synchronous somatic embryogenesis system in carrot suspension cultures. Using this system, we investigated expression of organ and embryo specific genes during embryogenesis. One of them, CEM6 was expressed specifically in the globular stage and can be considered as a really embryo specific gene which encodes a proline-rich protein containing signal peptides. The results obtained will be discussed in relation to expression of totipotency. *Present address: Japan Women's University, Tokyo 112 Japan Gene expression in the cell cycle of Catharanthus roseus cells -193-

5 Regulation and Manipulation of Pigment Colour in Flowers J. Mol, F. Quattrocchio, J. Wing, R. Koes, D. Weiss, L. Mur, C. Spelt, R. v. Blokland, P. de Lange, M. Stam, M. Kreike and J. Kooter. Free University, Department of Genetics, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. Flavonoids constitute a family of secondary metabolites common to all higher plants. They are synthesised via a branched pathway yielding different sub-classes of flavonoids, each serving a variety of functions in plant development and reproduction (see Figure). The red and purple anthocyanin pigments, as well as the colourless flavonols and flavanones found in flower petals of many plant species, serve as attractants for pollinating insects. Plant species that are self-pollinating (e.g. Arabidopsis) or are pollinated by wind (e.g. maize) also accumulate anthocyanins in a variety of tissues, indicating that anthocyanins may have additional functions. Flavonols accumulating in leaves are thought to act as UV protectants, whereas in anthers they are required for the development of the pollen cells. In leguminous plants, flavonols play a crucial role as signal molecules in the interaction with nodulating microbes and the defence against phytopathogens. In petunia plants the genes and and anll control synthesis of flavonoid pigments in the limb and tube of the flower corolla, the anthers and the seed coat. The an2 and an4 genes in contrast control only pigmentation of the corolla limb and anthers respectively. Analyses of steady-state and nascent mrna levels demonstrated that these four genes are regulatory genes involved in the transcriptional control of genes, encoding enzymes from the flavonoid pathway. Transient expression analyses in which a!3-glucuronidase reporter gene driven by the dihydroflavonol 4-reductase promoter (dfra-gus) was delivered into floral tissues via particle bombardment, showed that its expression is down-regulated in and, an2- and and l - flower corollas. Complementation experiments, in which dfra-gus was co-delivered with gene constructs expressing the maize regulatory pigmentation genes Lc or Cl into mutant flowers, indicated that an2 and an4 are members of a gene family functionally related to Cl. Similar experiments showed that expression of Lc and Cl is both necessary and sufficient for activation of the dfra promoter in leaf tissue indicating that the homologous petunia genes are important factors for establishing flower-specific expression of the structural flavonoid genes. Despite the functional similarity of regulatory pigmentation genes from maize and petunia they control a different set of target genes. Based on these findings we developed a model for the evolution of the flavonoid biosynthetic pathway and its regulators. In Petunia hybrida flavonoid gene expression is induced by gibberellic acid. I will discuss the effects of gibberellic acid, abscisic acid, auxins and cytokinins on the regulation of pigment biosynthesis in Petunia. Expression of antisense genes specifically suppresses the expression of the homologous endogenous gene which has been shown to offer a valuable tool to study gene function and biochemical processes. We employed antisense genes to block the synthesis of flavonoids which are the precursors of anthocyanin pigments. The constitutive expression of antisense chalcone synthase (chs) cdna in petunia reduced chs mrna levels in flowers and altered the pigmentation. The majority of the plants produced corollas with fully pigmented sectors flanked by white sectors. The pigmented area becomes larger when the plants are exposed to high intensity light or when they are sprayed with the phytohormone gibberellic acid.

6 Figure (.Schematic representation of the flavonoid biosynthesis pathway in petunia and maize. In the middle the pathway enzymes are arranged in chronological order (from top to bottom). On the immediate left and right the genetic loci corresponding to the enzyme genes are depicted. CHS, chalcone synthase: CHI, chalcone isomerasc: F3H, tlavanone3-hydroxylase: DFR, dihydrotlavonol 4-reductase; AS. anthocyanin synthase; 3GT, 3-glycosyl transferase: Rt, rutinosyl transferase: 5GT, 5-glycosyl transferase: MT/MF, methyltransferases. On the outside the factors regulating the pathway are shown. The transcription of both the endogenous and antisense transgenes was the same in the pigmented and in the white sectors which suggests that antisense RNA is necessary but not sufficient for gene suppression. The other factors that determine the efficiency by which antisense RNAs work remain to be determined. Transgenic petunias that contain one or more extra chs genes which were introduced as transgenes produce flowers with similar phenotypes as the antisense chs plants. This phenomenon is called sensesuppression. It shares many features with the antisense gene-induced phenotypes, including the sensitivity to light and gibberellic acid, which suggests that the mechanism(s) have elements in common. Nuclear run-on transcription assays with isolated nuclei showed that sense-suppression is also a post-transcriptional process which is influenced by endogenous and external factors. The possible role of antisense RNA in sense-suppression will be discussed.

7 Applying Large-Scale Methods to the Small Genome of Arabidopsis C.R. Somerville, T. Newman, J. Browse, V.Arondel, S. Gibson, K. Iba, DOE Plant Research Laboratory, Michigan State University, East Lansing, MI USA The growth of interest in Arabidopsis as a model organism for plant genetics was originally stimulated by the potential technical advantages associated with the unusually small genome size. However, it is only recently that the small genome size has begun to be exploited. The development of yeast artificial chromosome (YAC) libraries and high density RFLP or RAPD maps has permitted the map-based cloning of several genes. In principle, it is now possible to clone any Arabidopsis gene for which genetic variation can be genetically mapped. Thus, the development of criteria for the isolation of informative mutants has assumed a new importance. Recent improvements in methods for identifying and mapping DNA sequence polymorphism promise to greatly facilitate the further development and application of these methods. Also, the pending completion of maps of overlapping YACs will greatly facilitate the map-based cloning of genes. A parallel development which promises to facilitate the isolation and analysis of plant genes is large-scale cdna sequencing. Several groups in the world, including our own, have initiated the partial sequencing of all the cdna clones in Arabidopsis. The sequence of approximately 400 nucleotides from the 5' end of approximately 2500 randomly chosen clones has been completed. This resulted in the probable identification of approximately 600 genes. Thus, this approach is an unusually productive mechanism for exploiting the wealth of sequence information available in other organisms for the identification of plant genes.