Isotopically nonstationary metabolic flux analysis of plants: recent progress and future opportunities.

Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST-MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast-growing algae, and produced a synergistic multi-organ flux map that is a first in MFA for any biological system. These insights could not be elucidated through other approaches and show the potential of INST-MFA to correct an oversimplified understanding of plant metabolism.

SIMPEL: using stable isotopes to elucidate dynamics of context specific metabolism.

The capacity to leverage high resolution mass spectrometry (HRMS) with transient isotope labeling experiments is an untapped opportunity to derive insights on context-specific metabolism, that is difficult to assess quantitatively. Tools are needed to comprehensively mine isotopologue information in an automated, high-throughput way without errors. We describe a tool, Stable Isotope-assisted Metabolomics for Pathway Elucidation (SIMPEL), to simplify analysis and interpretation of isotope-enriched HRMS datasets. The efficacy of SIMPEL is demonstrated through examples of central carbon and lipid metabolism. In the first description, a dual-isotope labeling experiment is paired with SIMPEL and isotopically nonstationary metabolic flux analysis (INST-MFA) to resolve fluxes in central metabolism that would be otherwise challenging to quantify. In the second example, SIMPEL was paired with HRMS-based lipidomics data to describe lipid metabolism based on a single labeling experiment. Available as an R package, SIMPEL extends metabolomics analyses to include isotopologue signatures necessary to quantify metabolic flux.

Physcomitrium patens response to elevated CO2 is flexible and determined by an interaction between sugar and nitrogen availability.

Mosses hold a unique position in plant evolution and are crucial for protecting natural, long-term carbon storage systems such as permafrost and bogs. Due to small stature, mosses grow close to the soil surface and are exposed to high levels of CO2 , produced by soil respiration. However, the impact of elevated CO2 (eCO2 ) levels on mosses remains underexplored. We determined the growth responses of the moss Physcomitrium patens to eCO2 in combination with different nitrogen levels and characterized the underlying physiological and metabolic changes. Three distinct growth characteristics, an early transition to caulonema, the development of longer, highly pigmented rhizoids, and increased biomass, define the phenotypic responses of P. patens to eCO2 . Elevated CO2 impacts growth by enhancing the level of a sugar signaling metabolite, T6P. The quantity and form of nitrogen source influences these metabolic and phenotypic changes. Under eCO2 , P. patens exhibits a diffused growth pattern in the presence of nitrate, but ammonium supplementation results in dense growth with tall gametophores, demonstrating high phenotypic plasticity under different environments. These results provide a framework for comparing the eCO2 responses of P. patens with other plant groups and provide crucial insights into moss growth that may benefit climate change models.

Electric-field-induced colour switching in colloidal quantum dot molecules at room temperature.

Colloidal semiconductor quantum dots are robust emitters implemented in numerous prototype and commercial optoelectronic devices. However, active fluorescence colour tuning, achieved so far by electric-field-induced Stark effect, has been limited to a small spectral range, and accompanied by intensity reduction due to the electron-hole charge separation effect. Utilizing quantum dot molecules that manifest two coupled emission centres, we present a unique electric-field-induced instantaneous colour-switching effect. Reversible emission colour switching without intensity loss is achieved on a single-particle level, as corroborated by correlated electron microscopy imaging. Simulations establish that this is due to the electron wavefunction toggling between the two centres, induced by the electric field, and affected by the coupling strength. Quantum dot molecules manifesting two coupled emission centres may be tailored to emit distinct colours, opening the path for sensitive field sensing and colour-switchable devices such as a novel pixel design for displays or an electric-field-induced colour-tunable single-photon source.

Metabolic synergy in Camelina reproductive tissues for seed development.

Photosynthesis in fruits is well documented, but its contribution to seed development and yield remains largely unquantified. In oilseeds, the pods are green and elevated with direct access to sunlight. With 13C labeling in planta and through an intact pod labeling system, a unique multi-tissue comprehensive flux model mechanistically described how pods assimilate up to one-half (33 to 45%) of seed carbon by proximal photosynthesis in Camelina sativa. By capturing integrated tissue metabolism, the studies reveal the contribution of plant architecture beyond leaves, to enable seed filling and maximize the number of viable seeds. The latent capacity of the pod wall in the absence of leaves contributes approximately 79% of seed biomass, supporting greater seed sink capacity and higher theoretical yields that suggest an opportunity for crop productivity gains.

An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids.

Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.

Suppression of SDP1 Improves Soybean Seed Composition by Increasing Oil and Reducing Undigestible Oligosaccharides.

In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase. Three soybean genes orthologous to the Arabidopsis lipase gene SUGAR-DEPENDENT1 (SDP1) are upregulated at this time. Suppression of these genes resulted in higher oil levels, with lipid levels in the best lines exceeding 24% of seed weight. In addition, lipase-suppressed lines produced larger seeds compared to wild-type plants, resulting in increases of over 20% in total lipid per seed. Levels of raffinose and stachyose were lower in the transgenic lines, with average reductions of 15% in total raffinose family oligosaccharides observed. Despite the increase in oil, protein content was not negatively impacted and trended higher in the transgenic lines. These results are consistent with a role for SDP1 in turning over TAG to supply carbon for other needs, including the synthesis of oligosaccharides, and offer new strategies to further improve the composition of soybean seeds.

Complete Mapping of Interacting Charging States in Single Coupled Colloidal Quantum Dot Molecules.

Colloidal quantum dots (CQDs), major building blocks in modern optoelectronic devices, have so far been synthesized with only one emission center where the exciton resides. Recent development of coupled colloidal quantum dots molecules (CQDM), where two core-shell CQDs are fused to form two emission centers in close proximity, allows exploration of how charge carriers in one CQD affect the charge carriers in the other CQD. Cryogenic single particle spectroscopy reveals that while CQD monomers manifest a simple emission spectrum comprising a main emission peak with well-defined phonon sidebands, CQDMs exhibit a complex spectrum with multiple peaks that are not all spaced according to the known phonon frequencies. Based on complementary emission polarization and time-resolved analysis, this is assigned to fluorescence of the two coupled emission centers. Moreover, the complex peak structure shows correlated spectral diffusion indicative of the coupling between the two emission centers. Utilizing Schrödinger-Poisson self-consistent calculations, we directly map the spectral behavior, alternating between neutral and charged states of the CQDM. Spectral shifts related to electrostatic interaction between a charged emission center and the second emission center are thus fully mapped. Furthermore, effects of moving surface charges are identified, whereby the emission center proximal to the charge shows larger shifts. Instances where the two emission centers are negatively charged simultaneously are also identified. Such detailed mapping of charging states is enabled by the coupling within the CQDM and its anisotropic structure. This understanding of the coupling interactions is progress toward quantum technology and sensing applications based on CQDMs.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Neck Barrier Engineering in Quantum Dot Dimer Molecules via Intraparticle Ripening.

Coupled colloidal quantum dot (CQD) dimers represent a new class of artificial molecules composed of fused core/shell semiconductor nanocrystals. The electronic coupling and wave function hybridization are enabled by the formation of an epitaxial connection with a coherent lattice between the shells of the two neighboring quantum dots where the shell material and its dimensions dictate the quantum barrier characteristics for the charge carriers. Herein we introduce a colloidal approach to control the neck formation at the interface between the two CQDs in such artificial molecular constructs. This allows the tailoring of the neck barrier in prelinked homodimers formed via fusion of multifaceted wurtzite CdSe/CdS CQDs. The effects of reaction time, temperature, and excess ligands are studied. The neck filling process follows an intraparticle ripening mechanism at relatively mild reaction conditions while avoiding interparticle ripening. The degree of surface ligand passivation plays a key role in activating the surface atom diffusion to the neck region. The degree of neck filling strongly depends also on the initial relative orientation of the two CQDs, where homonymous plane attachment allows for facile neck growth, unlike the case of heteronymous plane attachment. Upon neck filling, the observed red-shift of the absorption and fluorescence measured both for ensemble and single dimers is assigned to enhanced hybridization of the confined wave function in CQD dimer molecules, as supported by quantum calculations. The fine-tuning of the particle interface introduced herein provides therefore a powerful tool to further control the extent of hybridization and coupling in CQD molecules.

Luminescent Anisotropic Wurtzite InP Nanocrystals.

Indium phosphide (InP) nanocrystals are emerging as an alternative to heavy metal containing nanocrystals for optoelectronic applications but lag behind in terms of synthetic control. Herein, luminescent wurtzite InP nanocrystals with narrow size distribution were synthesized via a cation exchange reaction from hexagonal Cu3P nanocrystals. A comprehensive surface treatment with NOBF4 was performed, which removes excess copper while generating stoichiometric In/P nanocrystals with fluoride surface passivation. The attained InP nanocrystals manifest a highly resolved absorption spectrum with a narrow emission line of 80 meV, and photoluminescence quantum yield of up to 40%. Optical anisotropy measurements on ensemble and single particle bases show the occurrence of polarized transitions directly mirroring the anisotropic wurtzite lattice, as also manifested from modeling of the quantum confined electronic levels. This shows a green synthesis path for achieving wurtzite InP nanocrystals with desired optoelectronic properties including color purity and light polarization with potential for diverse optoelectronic applications.

Semiconductor Bow-Tie Nanoantenna from Coupled Colloidal Quantum Dot Molecules.

Top-down fabricated nanoantenna architectures of both metallic and dielectric materials show powerful functionalities for Raman and fluorescence enhancement with relevance to single molecule sensing while inducing directionality of chromophore emission with implications for single photon sources. We synthesize the smallest bow-tie nanoantenna by selective tip-to-tip fusion of two tetrahedral colloidal quantum dots (CQDs) forming a dimer. While the tetrahedral monomers emit non-polarized light, the bow-tie architecture manifests nanoantenna functionality of enhanced emission polarization along the bow-tie axis, as predicted theoretically and revealed by single-particle spectroscopy. Theory also predicts the formation of an electric-field hotspot at the bow-tie epicenter. This is utilized for selective light-induced photocatalytic metal growth at that location, unlike growth on the free tips in dark conditions, thus demonstrating bow-tie dimer functionality as a photochemical reaction center.

Analyzing Mass Spectrometry Imaging Data of 13C-Labeled Phospholipids in Camelina sativa and Thlaspi arvense (Pennycress) Embryos.

The combination of 13C-isotopic labeling and mass spectrometry imaging (MSI) offers an approach to analyze metabolic flux in situ. However, combining isotopic labeling and MSI presents technical challenges ranging from sample preparation, label incorporation, data collection, and analysis. Isotopic labeling and MSI individually create large, complex data sets, and this is compounded when both methods are combined. Therefore, analyzing isotopically labeled MSI data requires streamlined procedures to support biologically meaningful interpretations. Using currently available software and techniques, here we describe a workflow to analyze 13C-labeled isotopologues of the membrane lipid and storage oil lipid intermediate-phosphatidylcholine (PC). Our results with embryos of the oilseed crops, Camelina sativa and Thlaspi arvense (pennycress), demonstrated greater 13C-isotopic labeling in the cotyledons of developing embryos compared with the embryonic axis. Greater isotopic enrichment in PC molecular species with more saturated and longer chain fatty acids suggest different flux patterns related to fatty acid desaturation and elongation pathways. The ability to evaluate MSI data of isotopically labeled plant embryos will facilitate the potential to investigate spatial aspects of metabolic flux in situ.

Coupled Colloidal Quantum Dot Molecules.

ConspectusElectronic coupling and hence hybridization of atoms serves as the basis for the rich properties for the endless library of naturally occurring molecules. Colloidal quantum dots (CQDs) manifesting quantum strong confinement possess atomic-like characteristics with s and p electronic levels, which popularized the notion of CQDs as artificial atoms. Continuing this analogy, when two atoms are close enough to form a molecule so that their orbitals start overlapping, the orbitals energies start to split into bonding and antibonding states made out of hybridized orbitals. The same concept is also applicable for two fused core-shell nanocrystals in close proximity. Their band edge states, which dictate the emitted photon energy, start to hybridize, changing their electronic and optical properties. Thus, an exciting direction of "artificial molecules" emerges, leading to a multitude of possibilities for creating a library of new hybrid nanostructures with novel optoelectronic properties with relevance toward diverse applications including quantum technologies.The controlled separation and the barrier height between two adjacent quantum dots are key variables for dictating the magnitude of the coupling energy of the confined wave functions. In the past, coupled double quantum dot architectures prepared by molecular beam epitaxy revealed a coupling energy of few millielectron volts, which limits the applications to mostly cryogenic operation. The realization of artificial quantum molecules with sufficient coupling energy detectable at room temperature calls for the use of colloidal semiconductor nanocrystal building blocks. Moreover, the tunable surface chemistry widely opens the predesigned attachment strategies as well as the solution processing ability of the prepared artificial molecules, making the colloidal nanocrystals as an ideal candidate for this purpose. Despite several approaches that demonstrated enabling of the coupled structures, a general and reproducible method applicable to a broad range of colloidal quantum materials is needed for systematic tailoring of the coupling strength based on a dictated barrierThis Account addresses the development of nanocrystal chemistry to create coupled colloidal quantum dot molecules and to study the controlled electronic coupling and their emergent properties. The simplest nanocrystal molecule, a homodimer formed from two core/shell nanocrystal monomers, in analogy to homonuclear diatomic molecules, serves as a model system. The shell material of the two CQDs is structurally fused, resulting in a continuous crystal. This lowers the potential energy barrier, enabling the hybridization of the electronic wave functions. The direct manifestation of the hybridization reflects on the band edge transition shifting toward lower energy and is clearly resolved at room temperature. The hybridization energy within the single homodimer molecule is strongly correlated with the extent of structural continuity, the delocalization of the exciton wave function, and the barrier thickness as calculated numerically. The hybridization impacts the emitted photon statistics manifesting faster radiative decay rate, photon bunching effect, and modified Auger recombination pathway compared to the monomer artificial atoms. Future perspectives for the nanocrystals chemistry paradigm are also highlighted.

ZnSe/ZnS Core/Shell Quantum Dots with Superior Optical Properties through Thermodynamic Shell Growth.

Epitaxial growth of a protective semiconductor shell on a colloidal quantum dot (QD) core is the key strategy for achieving high fluorescence quantum efficiency and essential stability for optoelectronic applications and biotagging with emissive QDs. Herein we investigate the effect of shell growth rate on the structure and optical properties in blue-emitting ZnSe/ZnS QDs with narrow emission line width. Tuning the precursor reactivity modifies the growth mode of ZnS shells on ZnSe cores transforming from kinetic (fast) to thermodynamic (slow) growth regimes. In the thermodynamic growth regime, enhanced fluorescence quantum yields and reduced on-off blinking are achieved. This high performance is ascribed to the effective avoidance of traps at the interface between the core and the shell, which are detrimental to the emission properties. Our study points to a general strategy to obtain high-quality core/shell QDs with enhanced optical properties through controlled reactivity yielding shell growth in the thermodynamic limit.

Colloidal quantum dot molecules manifesting quantum coupling at room temperature.

Coupling of atoms is the basis of chemistry, yielding the beauty and richness of molecules. We utilize semiconductor nanocrystals as artificial atoms to form nanocrystal molecules that are structurally and electronically coupled. CdSe/CdS core/shell nanocrystals are linked to form dimers which are then fused via constrained oriented attachment. The possible nanocrystal facets in which such fusion takes place are analyzed with atomic resolution revealing the distribution of possible crystal fusion scenarios. Coherent coupling and wave-function hybridization are manifested by a redshift of the band gap, in agreement with quantum mechanical simulations. Single nanoparticle spectroscopy unravels the attributes of coupled nanocrystal dimers related to the unique combination of quantum mechanical tunneling and energy transfer mechanisms. This sets the stage for nanocrystal chemistry to yield a diverse selection of coupled nanocrystal molecules constructed from controlled core/shell nanocrystal building blocks. These are of direct relevance for numerous applications in displays, sensing, biological tagging and emerging quantum technologies.

Electronic coupling in colloidal quantum dot molecules; the case of CdSe/CdS core/shell homodimers.

Coupled colloidal quantum dot molecules composed of two fused CdSe/CdS core/shell sphere monomers were recently presented. Upon fusion, the potential energy landscape changes into two quantum dots separated by a pretuned potential barrier with energetics dictated by the conduction and valence band offsets of the core/shell semiconductors and the width controlled by the shell thickness and the fusion reaction conditions. In close proximity of the two nanocrystals, orbital hybridization occurs, forming bonding and antibonding states in analogy to the hydrogen molecule. In this study, we examine theoretically the electronic and optical signatures of such a quantum dot dimer compared to its monomer core/shell building-blocks. We examine the effects of different core sizes, barrier widths, different band offsets, and neck sizes at the interface of the fused facets on the system wave-functions and energetics. Due to the higher effective mass of the hole and the large valence band offset, the hole still essentially resides in either of the cores, breaking the symmetry of the potential for the electron as well. We found that the dimer signature is well expressed in a red shift of the band gap both in absorption and emission, in slower radiative lifetimes and in an absorption cross section which is significantly enhanced relative to the monomers at energies above the shell absorption onset, while remains essentially at the same level near the band-edge. This study provides essential guidance to predesign of coupled quantum dot molecules with specific attributes which can be utilized for various new opto-electronic applications.

Metallic Conductive Luminescent Film.

We report a solution for the challenge of having luminescence and metal conductivity from the same material. The fabrication of a hybrid metal-conductive luminescent film that manifests this dual property is described: the conductivity arising from a continuous gold thin film structure and luminescence originating from the embedded fluorescent emitters (nanoparticles of silica-coated CdSe/CdS quantum dots (QD/SiO2 NPs)). The embedding of the QD/SiO2 NPs is performed via a self-templating gold electroless process. The presence of the insulating silica layer on the QDs avoids quenching and enables luminescence, while still allowing plasmonic coupling of the QDs, as observed by luminescence lifetime analysis and by surface-enhanced Raman scattering. The potential applications of this special dual functionality are demonstrated by its used as a temperature probe: Passing current (heating the gold thin film) affects the emission intensity and induces a spectral red-shift of the QD/SiO2 NPs. All properties of this metal-conductive luminescent film required the special embedding architecture and are not observed with simple adsorption of QD/SiO2 NPs on a continuous Au film.

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

BACKGROUND: Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state 13C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady state. RESULTS: We developed an in vitro plant culture strategy by using peppermint as a model plant with minimizing unlabelled carbon fixation where growing shoot tip was strongly dependent on labelled glucose for their carbon necessity. We optimized the light condition and detected the satisfactory phenotypical growth under the lower light intensity. Total volatile terpenes were also highest at the same light. Analysis of label incorporation into pulegone monoterpene after continuous U-13C6 glucose feeding revealed nearly 100% 13C, even at 15 days after first leaf visibility (DALV). Label enrichment was gradually scrambled with increasing light intensity and leaf age. This study was validated by showing similar levels of label enrichment in proteinogenic amino acids. The efficiency of this method was also verified in oregano. CONCLUSIONS: Our shoot tip culture depicted a method in achieving long term, stable and a high percentage of label accumulation in secondary metabolites within a fully functional growing plant system. It recommends the potential application for the investigations of various facets of plant metabolism by steady state 13C-MFA. The system also provides a greater potential to study sink leaf metabolism. Overall, we propose a system to accurately describe complex metabolic phenotypes in a growing plant.

Ground-State Heterogeneity along with Fluorescent Byproducts Causes Excitation-Dependent Fluorescence and Time-Dependent Spectral Migration in Citric Acid-Derived Carbon Dots.

The integrity of fluorescent carbon dot (FCD) emission deserves its highest appreciation when sample purification is performed with extreme care. Several controversial phenomena of FCD fluorescence including excitation-dependent emission, spectral migration with time, and thereby violation of the Kasha-Vavilov rule, which sparked intense debate during recent reports, disappeared when we rigorously purified the as-synthesized FCD sample. Purification was performed by first visual silica column chromatography (observing the emissions under UV illumination) and subsequently prolonged membrane dialysis. Most of the surprising phenomena of FCD fluorescence reported earlier apparently arose from ground-state spectral heterogeneity of FCD sample containing a large amount of fluorescent impurities (mostly polymeric or oligomeric in nature). Observation of our ensemble spectroscopic measurements, albeit nicely matched with recent reports based on single-particle experiments, differed largely from that of other ensemble measurements. Our results reconciled a number of long-standing controversies on FCD emission mostly by emphasizing the urgency of sample purification with more scientific rigor.