Helle Juel Martens

Quantification of Plasmodesmatal Endoplasmic Reticulum Coupling between Sieve Elements and Companion Cells Using Fluorescence Redistribution after Photobleaching

Transgenic tobacco (Nicotiana tabacum) was studied to localize the activity of phloem loading during development and to establish whether the endoplasmic reticulum (ER) of the companion cell (CC) and the sieve element (SE) reticulum is continuous by using a SUC2 promoter-green fluorescent protein (GFP) construct targeted to the CC-ER. Expression of GFP marked the collection phloem in source leaves and cotyledons as expected, but also the transport phloem in stems, petioles, midveins of sink leaves, nonphotosynthetic flower parts, roots, and newly germinated seedlings, suggesting that sucrose retrieval along the pathway is an integral component of phloem function. GFP fluorescence was limited to CCs where it was visualized as a well-developed ER network in close proximity to the plasma membrane. ER coupling between CC and SEs was tested in wild-type tobacco using an ER-specific fluorochrome and fluorescence redistribution after photobleaching (FRAP), and showed that the ER is continuous via pore-plasmodesma units. ER coupling between CC and SE was quantified by determining the mobile fraction and half-life of fluorescence redistribution and compared with that of other cell types. In all tissues, fluorescence recovered slowly when it was rate limited by plasmodesmata, contrasting with fast intracellular FRAP. FRAP was unaffected by treatment with cytochalasin D. The highest degree of ER coupling was measured between CC and SE. Intimate ER coupling is consistent with a possible role for ER in membrane protein and signal exchange between CC and SE. However, a complete lack of GFP transfer between CC and SE indicated that the intraluminal pore-plasmodesma contact has a size exclusion limit below 27 kD.

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

The first two steps of the biosynthesis of forskolin are active in Coleus forskohlii root cork cells harboring hydrophobic intracellular compartments used for terpenoid storage. Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.

Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum

Metabolite channeling by a dynamic metabolon The specialized metabolite dhurrin breaks down into cyanide when plant cell walls have been chewed, deterring insect pests. Laursen et al. found that the enzymes that synthesize dhurrin in sorghum assemble as a metabolon in lipid membranes (see the Perspective by Dsatmaichi and Facchini). The dynamic nature of metabolon assembly and disassembly provides dhurrin on an as-needed basis. Membrane-anchored cytochrome P450s cooperated with a soluble glucosyltransferase to channel intermediates toward efficient dhurrin production. Science, this issue p. 890; see also p. 829 Enzymes that synthesize a specialized metabolite congregate and disperse on an as-needed basis in the lipid membrane. Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.

Structural and Functional Vein Maturation in Developing Tobacco Leaves in Relation to AtSUC2 Promoter Activity

Transgenic tobacco (Nicotiana tabacum) plants expressing green fluorescent protein (GFP) from theAtSUC2 promoter were used to study the function of different vein classes in developing leaves. In sink leaves, unloading capacity occurred acropetally, with the class I (midrib) and class II veins becoming functional in phloem unloading before the maturation of the class III veinal network. In contrast, in developing cotyledons and source leaves, loading capacity occurred in a basipetal direction. There was a strong correlation between loading capacity, as assessed by14C Suc uptake and companion cell expression of AtSUC2-GFP. Developing cotyledons were shown to utilize all available vein classes for loading. A second line of transgenic plants was produced in which GFP, expressed from theAtSUC2 promoter, was targeted to the endoplasmic reticulum instead of the cytoplasm. In these AtSUC2-GFP-ER plants, GFP was unable to traffic into the sieve element and was restricted solely to the companion cells of source leaf tissues. Partial shading of leaves undergoing the sink-source transition demonstrated that the activation of the AtSUC2 promoter in tobacco was influenced by light. Functional and structural maturation of the minor veins required light or a product of light. The activation of theAtSUC2 promoter within major veins appears to be regulated differently from that in the minor veins. The relationship between AtSUC2 activation and the activity of endogenous tobacco Suc transporters is discussed.

Starch bioengineering affects cereal grain germination and seedling establishment

Summary Grain starch phosphorylation and amylose content affect germination and seedling establishment through the combination of direct effects on altered starch granule and molecular structure and indirect effects on amylase activities.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

Highlight A pathway containing two cytochrome P450s and a glucosyltransferase has been stably expressed in Nicotiana tabacum chloroplasts. The functional P450s are enriched in the thylakoids and receive electrons from photo-reduced ferredoxin.

Caged probes: a novel tool in studying symplasmic transport in plant tissues

Summary.Caged probes offer a novel approach to study plant cell-to-cell communication. Instead of introducing fluorescent molecules into cells by microinjection, their caged counterparts can be preloaded into the tissue by diffusion. Following spatially controlled photoactivation, movement of the uncaged fluorochrome can be followed in time and direction by confocal laser scanning microscopy. In the onion bulb scale epidermis used as a model system, symplasmic transport of the tracer out of a target cell was followed. Transport via the symplasmic pathway was challenged by plasmolysing the tissue. The experiments confirmed the symplasmic nature of tracer transport.

SVR4 (suppressor of variegation 4) and SVR4-like: two proteins with a role in proper organization of the chloroplast genetic machinery

SUPPRESSOR OF VARIEGATION 4 (SVR4, also called MRL7) and its homolog SVR4-like (also called MRL7-Like) were originally identified as important proteins for proper function of the chloroplast in Arabidopsis. Both are nuclear-encoded chloroplast-located proteins, and knockout mutants of either gene result in seedling lethality. Transmission electron microscopy analysis revealed that chloroplast development is arrested at an early developmental stage in both mutants. Accordingly, in the mutant plants severely decreased levels of photosynthetic pigments as well as subunits of the photosynthetic complexes could be detected. In absence of either of the two proteins chloroplast DNA organization was clearly affected. Immunological analysis revealed that SVR4 is a component of the transcriptionally active chromosome (TAC) from barley chloroplasts. Analyses of gene expression indicate that SVR4 and SVR4-like are required for proper function of the plastid transcriptional machinery. We propose that SVR4 and SVR4-like function as molecular chaperones ensuring proper organization of the nucleoids in chloroplasts.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Background: The plasma membrane H+-ATPase generates electrochemical gradients in plants and fungi. The minimal subunit organization required for activity is not known. Results: We developed a protocol for reconstitution of active H+-ATPase in nanodiscs. Conclusion: The minimal functional unit of the H+-ATPase is a monomer. Significance: The plasma membrane H+-ATPase functions like well characterized cation pumping P-type ATPases. Plasma membrane H+-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H+-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H+-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.