Subhasish Chatterjee

Tissue- and Cell-Type Specific Transcriptome Profiling of Expanding Tomato Fruit Provides Insights into Metabolic and Regulatory Specialization and Cuticle Formation

This study uses laser capture microdissection coupled with pyrosequencing to characterize the cell- and tissue-type transcriptomes of the pericarp of expanding tomato fruits. This provides new insights into the spatial distribution of expression of structural and regulatory genes associated with many metabolic pathways, and a cuticle lining the inner pericarp surface is described. Tomato (Solanum lycopersicum) is the primary model for the study of fleshy fruits, and research in this species has elucidated many aspects of fruit physiology, development, and metabolism. However, most of these studies have involved homogenization of the fruit pericarp, with its many constituent cell types. Here, we describe the coupling of pyrosequencing technology with laser capture microdissection to characterize the transcriptomes of the five principal tissues of the pericarp from tomato fruits (outer and inner epidermal layers, collenchyma, parenchyma, and vascular tissues) at their maximal growth phase. A total of 20,976 high-quality expressed unigenes were identified, of which more than half were ubiquitous in their expression, while others were cell type specific or showed distinct expression patterns in specific tissues. The data provide new insights into the spatial distribution of many classes of regulatory and structural genes, including those involved in energy metabolism, source-sink relationships, secondary metabolite production, cell wall biology, and cuticle biogenesis. Finally, patterns of similar gene expression between tissues led to the characterization of a cuticle on the inner surface of the pericarp, demonstrating the utility of this approach as a platform for biological discovery.

Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants

The aerial epidermis of all land plants is covered with a hydrophobic cuticle that provides essential protection from desiccation, and so its evolution is believed to have been prerequisite for terrestrial colonization. A major structural component of apparently all plant cuticles is cutin, a polyester of hydroxy fatty acids; however, despite its ubiquity, the details of cutin polymeric structure and the mechanisms of its formation and remodeling are not well understood. We recently reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL-motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here, we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin synthases within the large GDSL superfamily. We demonstrate that members of this ancient and conserved family of cutin synthase-like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution-state NMR analysis indicates that CD1 catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross-linking, may require additional, as yet unknown, factors.

Lasing from InGaP quantum dots in a spin-coated flexible microcavity

We report the realization of a mechanically flexible microcavity laser emitting at 657 nm using spin coating. These optically pumped vertical cavity surface emitting lasers use InGaP colloidal quantum dots as the active medium and alternating polymer layers of different refractive indices as the Bragg mirrors. Results of photoluminescence measurements indicating enhancement in spontaneous emission are presented. We also demonstrate the possibility of peeling the device off the substrate yielding a flexible structure that can conform to any shape and whose emission spectra can be mechanically tuned. This new class of hybrid lasers combines advantages of organic and inorganic materials.

Investigating the distance limit of a metal nanoparticle based spectroscopic ruler

Conventional Förster resonance energy transfer (FRET) processes involving a pair of fluorophore and organic quencher are restricted to an upper distance limit of ~10 nm. The application of a metal nanoparticle as a quencher can overcome the distance barrier of the traditional FRET technique. However, no standard distance dependence of this resonance energy transfer (RET) process has been firmly established. We have investigated the nonradiative energy transfer process between an organic donor (fluorescein) and gold nanoparticle quencher connected by double stranded (ds) DNA. The quenching efficiency of the gold nanoparticle as a function of distance between the donor and acceptor was determined by time-resolved lifetime analyses of the donor. Our results showed a 1/d4 distance dependence for the RET process for longer distances (>10 nm) and 1/d6 distance dependence for shorter distances (<10 nm). Our results clearly indicate the applicability of metal nanoparticle based quenchers for studying systems that exceed the 10 nm FRET barrier.

Probing Y-shaped DNA structure with time-resolved FRET

Self-assembly based on nucleic acid systems has become highly attractive for bottom-up fabrication of programmable matter due to the highly selective molecular recognition property of biomolecules. In this context, Y-shaped DNA (Y-DNA) provides an effective building block for forming unique self-assembled large-scale architectures. The dimension and growth of the nano- and microstructures depend significantly on the configurational stability of Y-DNA as a building block. Here we present structural studies of Y-DNA systems using a time-resolved FRET (Förster resonance energy transfer) technique. A fluorophore (Alexa 488) and an acceptor (DABCYL) were placed at two different ends of Y-DNA, and the lifetime of the fluorophore was measured to probe the relative distance between the donor and acceptor. Our results confirmed different distances between the arms of the Y-DNA and highlighted the overall structural integrity of the Y-DNA system as a leading building block for molecular self-assembly. Temperature dependent lifetime measurements indicated configurational changes in the overall Y-DNA nanoarchitecture above 40 °C.

Mini-review: What nuclear magnetic resonance can tell us about protective tissues

The epidermis and periderm protect plants from water and solute loss, pathogen invasion, and UV radiation. The cell walls of these protective tissues deposit the insoluble lipid biopolyesters cutin and suberin, respectively. These biopolymers interact in turn with polysaccharides, waxes and aromatic compounds to create complex assemblies that are not yet well defined at the molecular level. Non-destructive approaches must be tailored to the insoluble and noncrystalline character of these assemblies to establish the polymer and inter-component interactions needed to create functional barriers and structural supports. In the present mini-review, we illustrate the contribution of solid-state NMR methodology to compare the architecture of intact fruit cuticular polymers in wild-type and single-gene mutant tomatoes. We also show the potential of NMR-based metabolomics to identify the soluble metabolites that contribute to barrier formation in different varieties of potato tubers. Finally, we outline the challenges of these spectroscopic approaches, which include limited spectral resolution in solid state, differential swelling capabilities in solution, and incomplete dissolution in ionic liquids. Given the many genetically modified plants with altered suberin and cutin polymers that are now available, NMR nonetheless offers a promising tool to gain molecular insight into the complexity of these protective materials.

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

Background: Melanin is a poorly understood fungal virulence factor. Results: 2D 13C-13C correlation solid-state nuclear magnetic resonance reveals the carbon-based molecular architecture of intact melanin pigment assemblies in Cryptococcus neoformans. Conclusion: Polysaccharide cell-wall components form a scaffold for layered deposition of aromatic-based pigment assemblies. Significance: Deciphering macromolecular interactions that drive melanin pigment assembly in fungal cell walls facilitates the development of drug delivery materials. Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D 13C-13C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.

Deconstructing a Plant Macromolecular Assembly: Chemical Architecture, Molecular Flexibility, And Mechanical Performance of Natural and Engineered Potato Suberins

Periderms present in plant barks are essential protective barriers to water diffusion, mechanical breakdown, and pathogenic invasion. They consist of densely packed layers of dead cells with cell walls that are embedded with suberin. Understanding the interplay of molecular structure, dynamics, and biomechanics in these cell wall-associated insoluble amorphous polymeric assemblies presents substantial investigative challenges. We report solid-state NMR coordinated with FT-IR and tensile strength measurements for periderms from native and wound-healing potatoes and from potatoes with genetically modified suberins. The analyses include the intact suberin aromatic–aliphatic polymer and cell-wall polysaccharides, previously reported soluble depolymerized transmethylation products, and undegraded residues including suberan. Wound-healing suberized potato cell walls, which are 2 orders of magnitude more permeable to water than native periderms, display a strikingly enhanced hydrophilic–hydrophobic balance, a degradation-resistant aromatic domain, and flexibility suggestive of an altered supramolecular organization in the periderm. Suppression of ferulate ester formation in suberin and associated wax remodels the periderm with more flexible aliphatic chains and abundant aromatic constituents that can resist transesterification, attenuates cooperative hydroxyfatty acid motions, and produces a mechanically compromised and highly water-permeable periderm.

Activation of melanin synthesis in Alternaria infectoria by antifungal drugs

ABSTRACT The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.

Structural Characterization of Melanin Pigments from Commercial Preparations of the Edible Mushroom Auricularia auricula.

Many of the most widely consumed edible mushrooms are pigmented, and these have been associated with some beneficial health effects. Nevertheless, the majority of the reported compounds associated with these desirable properties are non-pigmented. We have previously reported that melanin pigment from the edible mushroom Auricularia auricula can protect mice against ionizing radiation, although no physicochemical characterization was reported. Consequently, in this study we have characterized commercial A. auricula mushroom preparations for melanin content and carried out structural characterization of isolated insoluble melanin materials using a panel of sophisticated spectroscopic and physical/imaging techniques. Our results show that approximately 10% of the dry mass of A. auricula is melanin and that the pigment has physicochemical properties consistent with those of eumelanins, including hosting a stable free radical population. Electron microscopy studies show that melanin is associated with the mushroom cell wall in a manner similar to that of melanin from the model fungus C. neoformans. Elemental analysis of melanin indicated C, H, and N ratios consistent with 5,6-dihydroxyindole-2-carboxylic acid/5,6-dihydroxyindole and 1,8-dihydroxynaphthalene eumelanin. Validation of the identity of the isolated product as melanin was achieved by EPR analysis. A. auricula melanin manifested structural differences, relative to the C. neoformans melanin, with regard to the variable proportions of alkyl chains or oxygenated carbons. Given the necessity for new oral and inexpensive radioprotective materials coupled with the commercial availability of A. auricula mushrooms, this product may represent an excellent source of edible melanin.