Utilizing Synchrotron-Based X-ray Micro-Computed Tomography to Visualize the Microscopic Structure of Starch Hydrogels In Situ.

This study aimed to visualize the microstructures of starch hydrogels using synchrotron-based X-ray micro-computed tomography (µCT). Waxy maize starch (WMS, 3.3% amylose, db), pea starch (PS, 40.3% amylose), and high-amylose maize starch (HMS, 63.6% amylose) were cooked at 95 and 140 °C to prepare starch hydrogels. WMS and HMS failed to form a gel after 95 °C cooking and storage, while PS developed a firm gel. At 140 °C cooking, HMS of a high amylose nature was fully gelatinized and generated a rigid gel with the highest strength. Both scanning electron microscopy (SEM) and µCT revealed the unique structural features of various starch hydrogels/pastes prepared at different temperatures, which were greatly affected by the degree of swelling and dispersity of the starches. As a nondestructive method, µCT showed certain advantages over SEM, including minimal shrinkage of the hydrogels, relatively simple sample preparation, and allowing for three-dimensional reconstruction of the hydrogel microstructure. This study indicated that synchrotron-based µCT could be a useful technique in visualizing biopolymer-based hydrogels.

Characterization of spring and durum wheat using non-destructive synchrotron phase contrast X-ray microtomography during storage.

Post-harvest losses during cereal grain storage are a big concern in both developing and developed countries, where spring and durum wheat are staple food grains. Varieties under these classes behave differently under storage, which affects their end storage life. High resolution imaging data of dry as well as spoiled seed are not available for any class of wheat; therefore, an attempt was made to generate 3D data for better understanding of seed structure and changes due to spoilage. Six wheat varieties (3 varieties for each class of wheat) were stored for 5 week at 17% moisture content (wb) before scanning. Seeds were also stored in a freezer (-18 °C) for further scanning to determine if any changes occur in the structure of seeds due to freezing. Spring varieties of wheat performed better than durum varieties and freezing did not affect seed structure. Data could also help plant breeders to develop varieties that do not easily spoil, adjust grain processing techniques, and develop post-harvest recommendations for other wheat varieties.

Nutri-cereal tissue-specific transcriptome atlas during development: Functional integration of gene expression to identify mineral uptake pathways in little millet (Panicum sumatrense).

Little millet (Panicum sumatrense Roth ex Roem. & Schult.) is an essential minor millet of southeast Asia and Africa's temperate and subtropical regions. The plant is stress-tolerant, has a short life cycle, and has a mineral-rich nutritional profile associated with unique health benefits. We report the developmental gene expression atlas of little millet (genotype JK-8) from ten tissues representing different stages of its life cycle, starting from seed germination and vegetative growth to panicle maturation. The developmental transcriptome atlas led to the identification of 342 827 transcripts. The BUSCO analysis and comparison with the transcriptomes of related species confirm that this study presents high-quality, in-depth coverage of the little millet transcriptome. In addition, the eFP browser generated here has a user-friendly interface, allowing interactive visualizations of tissue-specific gene expression. Using these data, we identified transcripts, the orthologs of which in Arabidopsis and rice are involved in nutrient acquisition, transport, and response pathways. The comparative analysis of the expression levels of these transcripts holds great potential for enhancing the mineral content in crops, particularly zinc and iron, to address the issue of "hidden hunger" and to attain nutritional security, making it a valuable asset for translational research.

The hidden diversity of vascular patterns in flower heads.

Vascular systems are intimately related to the shape and spatial arrangement of the plant organs they support. We investigate the largely unexplored association between spiral phyllotaxis and the vascular system in Asteraceae flower heads. We imaged heads of eight species using synchrotron-based X-ray micro-computed tomography and applied original virtual reality and haptic software to explore head vasculature in three dimensions. We then constructed a computational model to infer a plausible patterning mechanism. The vascular system in the head of the model plant Gerbera hybrida is qualitatively different from those of Bellis perennis and Helianthus annuus, characterized previously. Cirsium vulgare, Craspedia globosa, Echinacea purpurea, Echinops bannaticus, and Tanacetum vulgare represent variants of the Bellis and Helianthus systems. In each species, the layout of the main strands is stereotypical, but details vary. The observed vascular patterns can be generated by a common computational model with different parameter values. In spite of the observed differences of vascular systems in heads, they may be produced by a conserved mechanism. The diversity and irregularities of vasculature stand in contrast with the relative uniformity and regularity of phyllotactic patterns, confirming that phyllotaxis in heads is not driven by the vasculature.

Study of Microstructural, Nutritional, and Biochemical Changes in Hulled and Hulless Barley during Storage Using X-ray and Infrared Techniques.

Four varieties of barley (Esma, AC Metacalf, Tradition, and AB Cattlelac), representing four Canadian barley classes, were stored at 17% moisture content (mc) for 8 week. Stored barely was characterized using synchrotron X-ray phase contrast microcomputed tomography, synchrotron X-ray fluorescence imaging, and mid-infrared spectroscopy at the Canadian Light Source, Saskatoon. The deterioration was observed in all the selected varieties of barley at the end of 8 week of storage. Changes due to spoilage over time were observed in the grain microstructure and its nutrient distribution and composition. This study underscores the critical importance of the initial condition of barley grain microstructure in determining its storage life, particularly under unfavorable conditions. The hulled barley varieties showed more deterioration in microstructure than the hulless varieties of barley, where a direct correlation between microstructural changes and alterations in nutritional content was found. All selected barley classes showed changes in the distribution of nutrients (Ca, Fe, K, Mn, Cu, and Zn), but the two-row AC Metcalf variety exhibited more substantial variations in their nutrient distribution (Zn and Mn) than the other three varieties during storage. The two-row class barley varieties showed more changes in biochemical components (protein, lipids, and carbohydrates) than the six-row class varieties.

Drought-induced fiber water release and xylem embolism susceptibility of intact balsam poplar saplings.

Balsam poplar (Populus balsamifera L.) is a widespread tree species in North America with significant ecological and economic value. However, little is known about the susceptibility of saplings to drought-induced embolism and its link to water release from surrounding xylem fibers. Questions remain regarding localized mechanisms that contribute to the survival of saplings in vivo of this species under drought. Using X-ray micro-computed tomography on intact saplings of genotypes Gillam-5 and Carnduff-9, we found that functional vessels are embedded in a matrix of water-filled fibers under well-watered conditions in both genotypes. However, water-depleted fibers started to appear under moderate drought stress while vessels remained water-filled in both genotypes. Drought-induced xylem embolism susceptibility was comparable between genotypes, and a greater frequency of smaller diameter vessels in GIL-5 did not increase embolism resistance in this genotype. Despite having smaller vessels and a total vessel number that was comparable to CAR-9, stomatal conductance was generally higher in GIL-5 compared to CAR-9. In conclusion, our in vivo data on intact saplings indicate that differences in embolism susceptibility are negligible between GIL-5 and CAR-9, and that fiber water release should be considered as a mechanism that contributes to the maintenance of vessel functional status in saplings of balsam poplar experiencing their first drought event.

Synchrotron micro-computed tomography unveils the three-dimensional structure and origin of staminodes in the Plains Prickly Pear Cactus Opuntia polyacantha Haw. (Cactaceae).

Floral appendages display an array of shapes and sizes. Among these organs, staminodes are morphologically diverse structures that have lost the ability to produce pollen, but in some instances, they produce fertile pollen grains. In the family Cactaceae staminodes are uncommon and range from simple linear to flat to spatulate structures, but studies describing their structural attributes are scanty. This study highlights the advantages of synchrotron radiation for sample preparation and as a research tool for plant biology. It describes the internal morphology of floral parts, particularly stamen, tepal, and staminode in the Plains Prickly Pear Cactus, Opuntia polyacantha, using synchrotron radiation micro-computed tomography (SR-µCT). It also shows the different anatomical features in reconstructed three-dimensional imaging of reproductive parts and discuss the advantages of the segmentation method to detect and characterize the configuration and intricate patterns of vascular networks and associated structures of tepal and androecial parts applying SR-µCT. This powerful technology led to substantial improvements in terms of resolution allowing a more comprehensive understanding of the anatomical organization underlying the vasculature of floral parts and inception of staminodes in O. polyacantha. Tepal and androecial parts have uniseriate epidermis enclosing loose mesophyll with mucilage secretory ducts, lumen, and scattered vascular bundles. Cryptic underlying structural attributes provide evidence of a vascularized pseudo-anther conjoint with tepals. The undefined contours of staminodial appendages (pseudo-anther) amalgamated to the tepals' blurred boundaries suggest that staminodes originate from tepals, a developmental pattern supporting the fading border model of floral organ identity for angiosperms.

Decontamination of water co-polluted by copper, toluene and tetrahydrofuran using lauric acid.

Co-contamination by organic solvents (e.g., toluene and tetrahydrofuran) and metal ions (e.g., Cu2+) is common in industrial wastewater and in industrial sites. This manuscript describes the separation of THF from water in the absence of copper ions, as well as the treatment of water co-polluted with either THF and copper, or toluene and copper. Tetrahydrofuran (THF) and water are freely miscible in the absence of lauric acid. Lauric acid separates the two solvents, as demonstrated by proton nuclear magnetic resonance (1H NMR) and Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR). The purity of the water phase separated from 3:7 (v/v) THF:water mixtures using 1 M lauric acid is ≈87%v/v. Synchrotron small angle X-Ray scattering (SAXS) indicates that lauric acid forms reverse micelles in THF, which swell in the presence of water (to host water in their interior) and ultimately lead to two free phases: 1) THF-rich and 2) water-rich. Deprotonated lauric acid (laurate ions) also induces the migration of Cu2+ ions in either THF (following separation from water) or in toluene (immiscible in water), enabling their removal from water. Laurate ions and copper ions likely interact through physical interactions (e.g., electrostatic interactions) rather than chemical bonds, as shown by ATR-FTIR. Inductively coupled plasma-optical emission spectrometry (ICP-OES) demonstrates up to 60% removal of Cu2+ ions from water co-polluted by CuSO4 or CuCl2 and toluene. While lauric acid emulsifies water and toluene in the absence of copper ions, copper salts destabilize emulsions. This is beneficial, to avoid that copper ions are re-entrained in the water phase alongside with toluene, following their migration in the toluene phase. The effect of copper ions on emulsion stability is explained based on the decreased interfacial activity and compressional rigidity of interfacial films, probed using a Langmuir trough. In wastewater treatment, lauric acid (a powder) can be mixed directly in the polluted water. In the context of groundwater remediation, lauric acid can be solubilized in canola oil to enable its injection to treat aquifers co-polluted by organic solvents and Cu2+. In this application, injectable filters obtained by injecting cationic hydroxyethylcellulose (HEC +) would impede the flow of toluene and copper ions partitioned in it, protecting downstream receptors. Co-contaminants can be subsequently extracted upstream of the filters (using pumping wells), to enable their simultaneous removal from aquifers.

With a Little Help from My Cell Wall: Structural Modifications in Pectin May Play a Role to Overcome Both Dehydration Stress and Fungal Pathogens.

Cell wall structural modifications through pectin cross-linkages between calcium ions and/or boric acid may be key to mitigating dehydration stress and fungal pathogens. Water loss was profiled in a pure pectin system and in vivo. While calcium and boron reduced water loss in pure pectin standards, the impact on Allium species was insignificant (p > 0.05). Nevertheless, synchrotron X-ray microscopy showed the localization of exogenously applied calcium to the apoplast in the epidermal cells of Allium fistulosum. Exogenous calcium application increased viscosity and resistance to shear force in Allium fistulosum, suggesting the formation of calcium cross-linkages ("egg-box" structures). Moreover, Allium fistulosum (freezing tolerant) was also more tolerant to dehydration stress compared to Allium cepa (freezing sensitive). Furthermore, the addition of boric acid (H3BO3) to pure pectin reduced water loss and increased viscosity, which indicates the formation of RG-II dimers. The Arabidopsis boron transport mutant, bor1, expressed greater water loss and, based on the lesion area of leaf tissue, a greater susceptibility to Colletotrichum higginsianum and Botrytis cinerea. While pectin modifications in the cell wall are likely not the sole solution to dehydration and biotic stress resistance, they appear to play an important role against multiple stresses.

Tempering of cocoa butter and chocolate using minor lipidic components.

Chocolate manufacture includes a complex tempering procedure to direct the crystallization of cocoa butter towards the formation of fat crystal networks with specific polymorphism, nano- and microstructure, melting behavior, surface gloss and mechanical properties. Here we investigate the effects of adding various minor non-triglyceride lipidic components to refined cocoa butter and chocolate on their physical properties. We discover that addition of saturated phosphatidylcholine and phosphatidylethanolamine to neutralized and bleached cocoa butter or molten and recrystallized commercial chocolate at 0.1% (w/w) levels, followed by rapid cooling to 20 °C in the absence of shear, accelerates crystallization, stabilizes the desirable Form V polymorph and induces the formation of chocolate with an optimal microstructure, surface gloss and mechanical strength. Final chocolate structure and properties are comparable to those of a commercial tempered chocolate. Minor lipidic component addition represents an effective way to engineer chocolate material properties at different length scales, thus simplifying the entire tempering process.

Heat induced gelation of pulse protein networks.

This study examines the effect of salts (0.5 M NaCl or 0.25 M CaCl2) and protein concentration (7.5-15%) on the gel-forming abilities of lentil (LPC), yellow pea (YPC), and faba bean (FPC) protein concentrates formed at pH 7.0. The surface hydrophobicity of YPC (84.8 arbitrary units, a.u.) was found to be lower than LPC (147.2 a.u.) and FPC (135.0 a.u.). In contrast, the surface charge for LPC, YPC, and FPC was -37.8, -28.4, and -29.3 mV, respectively. The Lg/Vn ratio of YPCs was determined as 0.65 followed by LPC (0.57) and FPC (0.41). The presence of salts reduced the least gelling concentration. LPC and FPC also appeared to have a more ordered structure than YPC as evident by CLSM. The network appeared more ordered as the protein concentration increased or in the presence of NaCl or CaCl2 according to CLSM and synchrotron based micro computed tomography (µCT).

Phenotyping Plant Cellular and Tissue Level Responses to Cold with Synchrotron-Based Fourier-Transform Infrared Spectroscopy and X-Ray Computed Tomography.

Despite the extensive use of synchrotron radiation in material and biomedical sciences, it has only recently been utilized to expand our understanding of plant responses to environmental stress. Recent advances have led to the development of phenotyping platforms to identify chemical and morphological differences in breeding plant material. While these methodologies are applicable for and tested with a variety of abiotic and biotic stresses, they are particularly useful as a first step to identify cold-induced chemical and morphological changes in plants. Here, we describe two methods to determine cold acclimation-induced changes at the cellular and tissue levels. First, we illustrate how to quantify and visualize changes in tissue chemistry using Fourier-transform infrared spectroscopy. Second, we describe how to nondestructively prepare, analyze, and interpret X-ray phase contrast images and render this data as two- or three-dimensional models. While these techniques utilize synchrotron radiation, the methodology and standard practices are applicable for handheld and laboratory bench-top equipment operating with conventional light sources.

High throughput nutritional profiling of pea seeds using Fourier transform mid-infrared spectroscopy.

Seed samples from 117 genetically diverse pea breeding lines were used to determine the robustness of Fourier transform mid-infrared spectroscopy (FT-MIR) for the rapid nutritional profiling of seeds. The FT-MIR results were compared to wet chemistry methods for assessing the concentrations of total protein, starch, fiber, phytic acid, and carotenoids in pea seed samples. Of the five partial least square regression models (PLSR) developed, protein, fiber and phytic acid concentrations predicted by the models exhibited correlation coefficients greater than 0.83 when compared with data obtained using the wet chemistry methods for both the calibration and validation sets. The starch PLSR model had a correlation greater than 0.75, and carotenoids had correlation of 0.71 for the validation sets. The methods implemented in this research show the novelty and usefulness of FT-MIR as a simple, fast, and cost-effective technique to determine multiple seed constituents simultaneously.

Mid-infrared spectroscopy is a fast screening method for selecting Arabidopsis genotypes with altered leaf cuticular wax.

Arabidopsis eceriferum (cer) mutants with unique alterations in their rosette leaf cuticular wax accumulation and composition established by gas chromatography have been investigated using attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy in combination with univariate and multivariate analysis. Objectives of this study were to evaluate the utility of ATR-FTIR for detection of chemical diversity in leaf cuticles, obtain spectral profiles of cer mutants in comparison with the wild type, and identify changes in leaf cuticles caused by drought stress. FTIR spectra revealed both genotype- and treatment-dependent differences in the chemical make-up of Arabidopsis leaf cuticles. Drought stress caused specific changes in the integrated area of the CH3 peak, asymmetrical and symmetrical CH2 peaks, ester carbonyl peak and the peak area ratio of ester CO to CH2 asymmetrical vibration. CH3 peak positively correlated with the total wax accumulation. Thus, ATR-FTIR spectroscopy is a valuable tool that can advance our understanding of the role of cuticle chemistry in plant response to drought and allow selection of superior drought-tolerant varieties from large genetic resources.

Showcasing the application of synchrotron-based X-ray computed tomography in host-pathogen interactions: The role of wheat rachilla and rachis nodes in Type-II resistance to Fusarium graminearum.

Fusarium head blight, caused primarily by Fusarium graminearum (Fg), is one of the most devastating diseases of wheat. Host resistance in wheat is classified into five types (Type-I to Type-V), and a majority of moderately resistant genotypes carry Type-II resistance (resistance to pathogen spread in the rachis) alleles, mainly from the Chinese cultivar Sumai 3. Histopathological studies in the past failed to identify the key tissue in the spike conferring resistance to pathogen spread, and most of the studies used destructive techniques, potentially damaging the tissue(s) under study. In the present study, nondestructive synchrotron-based phase contrast X-ray imaging and computed tomography techniques were used to confirm the part of the wheat spike conferring Type-II resistance to Fg spread, thus showcasing the application of synchrotron-based techniques to image host-pathogen interactions. Seven wheat genotypes of moderate resistance to Fusarium head blight were studied for changes in the void space volume fraction and grayscale/voxel intensity following Fg inoculation. Cell-wall biopolymeric compounds were quantified using Fourier-transform midinfrared spectroscopy for all genotype-treatment combinations. The study revealed that the rachilla and rachis nodes together are structurally important in conferring Type-II resistance. The structural reinforcement was not necessarily observed from lignin deposition but rather from an unknown mechanism.