The genomic organization of two extracellular invertase genes from tomato (Lin5 and Lin7), which are linked in a direct tandem repeat, and their tissue-specific and hormone-inducible expression are shown. Transient expression analysis ofLin5 promoter sequences fused to the β-glucuronidase (GUS) reporter gene (uidA) demonstrates a specific expression of Lin5during tomato fruit development. A Lin5 promoter fragment was fused to the truncated nos promoter to analyse hormone induction via GUS reporter gene activity in transiently transformed tobacco leaves. A specific up-regulation of GUS activity conferred by this Lin5 promoter fragment in response to gibberellic acid (GA), auxin and abscisic acid (ABA) treatment was observed, indicating a critical role of the regulation of Lin5 by phytohormones in tomato flower and fruit development. In situ hybridization analysis of Lin7 shows a high tissue-specific expression in tapetum and pollen. These results support an important role for Lin5 and Lin7 extracellular invertases in the development of reproductive organs in tomato and contribute to unravel the underlying regulatory mechanisms.

Phenylphenalenones represent a typical group of secondary metabolites of the Haemodoraceae. Some of these phenolic compounds show organ-specific distribution within the plant. However, detailed information on cellular localisation is still lacking. To this end, confocal laser-scanning microscopy, microspectral photometry and high-performance liquid chromatography were used to study the tissue localisation of phenylphenalenone-type compounds in Xiphidium caeruleum Aubl. From the autofluorescence potential of these compounds, specific distribution of allophanylglucosides and non-glucosidic compounds of the phenylphenalenone-type in distinct cells of the roots (apical meristem, cortex, cap, epidermis) and the shoot system was revealed. Fluorescence enhancement using "Naturstoff reagent A" (NA) indicated the occurrence of NA-positive natural products in the vacuoles of leaf epidermal cells. The present results provide new insights into the possible functions of phenylphenalenone-related compounds in the context of their localisation. Additionally, the advantages and limitations of the techniques are discussed.

The most abundant jasmonate-induced protein (JIP) in barley leaves is a 23 kDa protein (JIP23). Its function, however, is unknown. In order to analyze its function by homologous transformation, new plasmid vectors have been constructed. They carry the cDNA coding for JIP23 in sense or antisense orientation under the control of the Ubi-1-promoter as well as the pat resistance gene under the control of the 35S promoter. Barley mesophyll protoplasts were transiently transformed with the sense constructs. PAT activity and immunological detection of JIP23 could be achieved in transformed protoplasts but not in untransformed protoplasts indicating that the construct was active. Thus, these new vectors are suitable for stable transformation of barley. Carrying a multiple cloning site (MCS), these vectors can be used now in a wide range of transformation of barley.

Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins).

The Arabidopsis genome contains a family of NDR1/HIN1-like (NHL) genes that show homology to the nonrace-specific disease resistance (NDR1) and the tobacco (Nicotiana tabacum) harpin-induced (HIN1) genes. NHL3 is a pathogen-responsive member of this NHL gene family that is potentially involved in defense. In independent transgenic NHL3-overexpressing plant lines, a clear correlation between increased resistance to virulent Pseudomonas syringae pv. tomato DC3000 and enhanced NHL3 transcript levels was seen. These transgenic plants did not show enhanced pathogenesis-related gene expression or reactive oxygen species accumulation. Biochemical and localization experiments were performed to assist elucidation of how NHL3 may confer enhanced disease resistance. Gene constructs expressing amino-terminal c-myc-tagged or carboxyl-terminal hemagglutinin epitope (HA)-tagged NHL3 demonstrated membrane localization in transiently transformed tobacco leaves. Stable Arabidopsis transformants containing the NHL3-HA construct corroborated the findings observed in tobacco. The detected immunoreactive proteins were 10 kD larger than the calculated size and could be partially accounted for by the glycosylation state. However, the expected size was not attained with deglycosylation, suggesting possibly additional posttranslational modification. Detergent treatment, but not chemicals used to strip membrane-associated proteins, could displace the immunoreactive signal from microsomal fractions, showing that NHL3 is tightly membrane associated. Furthermore, immunofluorescence and immunogold labeling, coupled with two-phase partitioning techniques, revealed plasma membrane localization of NHL3-HA. This subcellular localization of NHL3 positions it at an initial contact site to pathogens and may be important in facilitating interception of pathogen-derived signals.

Mycorrhizas are the most important mutualistic symbioses on earth. The most prevalent type are the arbuscular mycorrhizas (AMs) that develop between roots of most terrestrial plants and fungal species of the Zygomycota. The AM fungi are able to grow into the root cortex forming intercellular hyphae from which highly branched structures, arbuscules, originate within cortex cells. The arbuscules are responsible for nutrient exchange between the host and the symbiont, transporting carbohydrates from the plant to the fungus and mineral nutrients, especially phosphate, and water from the fungus to the plant. Plants adapt their phosphate uptake to the interaction with the AM fungus by synthesis of specific phosphate transporters. Colonization of root cells induces dramatic changes in the cytoplasmic organization: vacuole fragmentation, transformation of the plasma membrane to a periarbuscular membrane covering the arbuscule, increase of the cytoplasm volume and numbers of cell organelles, as well as movement of the nucleus into a central position. The plastids form a dense network covering the symbiotic interface. In some of these changes, microtubules are most likely involved. With regard to the molecular crosstalk between the two organisms, a number of phytohormones (cytokinins, abscisic acid, jasmonate) as well as various secondary metabolites have been examined: (i) Jasmonates occur at elevated level, which is accompanied by cell-specific expression of genes involved in jasmonate biosynthesis that might be linked to strong carbohydrate sink function of AM roots and induced defense reactions; (ii) apocarotenoids (derivatives of mycorradicin and glycosylated cyclohexenones) accumulate in most mycorrhizal roots examined so far. Their biosynthesis via the nonmevalonate methylerythritol phosphate (MEP) pathway has been studied resulting in new insights into AM-specific gene expression and biosynthesis of secondary isoprenoids.

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.

The allene oxide cyclase (AOC)‐catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue‐specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i) activation of systemin‐dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii) the tissue‐specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii) the tissue‐specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.

A Medicago truncatula cDNA encoding a germin-like protein (GLP) was isolated from a suppression subtractive hybridization cDNA library enriched for arbuscular mycorrhiza (AM)-induced genes. The MtGLP1 amino acid sequence shows some striking differences to previously described plant GLP sequences and might therefore represent a new subgroup of this multigene family. The MtGlp1 mRNA was strongly induced in roots and root cultures colonized by the AM fungus Glomus intraradices. Whereas MtGlp1 is strongly induced in AM, no transcripts of the gene were detected in non-infected roots or in roots after infection with the oomycete root pathogen Aphanomyces euteiches or with Rhizobia. Increased phosphate levels during fertilization also could not stimulate MtGlp1 transcription. Hence, MtGlp1 induction seems to be an AM-specific phenomenon. In situ hybridization showed that MtGlp1 is localized in arbuscule containing cells. A putative orthologue of this AM-specific GLP gene could be localized in a second legume Lotus japonicus, indicating that the regulation of a member of the GLP family belongs to a conserved mechanism in AM regulation in different plant species.

Occurrence of an essential enzyme in jasmonate (JA) biosynthesis, the allene oxide cyclase, (AOC) was analyzed in different developmental stages and various organs of Arabidopsis thaliana plants by immuno blot analysis and immunocytological approaches. Levels of AOC and of the two preceding enzymes in JA biosynthesis increased during seedling development accompanied by increased levels of JA and 12-oxophytodienoic acid levels after 4 and 8 weeks. Most tissues including all vascular bundles and that of flower buds contain AOC protein. Flowers shortly before opening, however, contain AOC protein preferentially in ovules, stigma cells and vascular bundles, whereas in anthers and pollen AOC could not be detected. The putative roles of AOC and JA in development are discussed.The allene oxide cyclase (AOC) is an important enzyme in jasmonate biosynthesis. Levels and occurrence of AOC in different organs and tissues are altered during development of Arabidopsis thaliana.