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.

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.

Identification of ultrastructural and biochemical cuticular markers influencing temperature of ice nucleation in selected genotypes of corn.

Corn is an economically important yet frost-sensitive crop, injured at the moment of ice nucleation. However, the influence of autumn temperatures on subsequent ice nucleation temperature is unknown. A 10-day chilling treatment under phytotron conditions ("mild", 18/6°C) or ("extreme", 10/5°C) generated no-visible damage but induced changes in the cuticle of the four genotypes in this study. The putatively more cold hardy Genotypes 884 and 959 leaves nucleated at colder temperatures compared to the more sensitive Genotypes 675 and 275. After chilling treatment, all four genotypes displayed warmer ice nucleation temperatures, with Genotype 884 expressing the largest shift to warmer nucleation temperatures. Cuticular hydrophobicity reduced while cuticular thickness remained unchanged under the chilling treatment. By contrast, under five-week field conditions, cuticle thickness increased in all genotypes, with Genotype 256 expressing a significantly thinner cuticle. FTIR spectroscopy revealed increases in the spectral regions of cuticular lipids in all genotypes after phytotron chilling treatment, while those spectral regions decreased under field conditions. A total of 142 molecular compounds were detected, with 28 compounds significantly induced under either phytotron or field conditions. Of these, seven compounds were induced under both conditions (Alkanes C31-C33, Ester C44, C46, ß-amyrin, and triterpene). While clear differential responses were observed, chilling conditions preceding a frost modified physical and biochemical properties of the leaf cuticle under both phytotron and field conditions indicating this response is dynamic and could be a factor in selecting corn genotypes better adapted to avoiding frost with lower ice nucleation temperature.

Non-destructive characterization of pulse flours-A review.

The consumption of plant-based proteins sourced from pulses is sustainable from the perspective of agriculture, environment, food security, and nutrition. Increased incorporation of high-quality pulse ingredients into foods such as pasta and baked goods is poised to produce refined food products to satisfy consumer demand. However, a better understanding of pulse milling processes is required to optimize the blending of pulse flours with wheat flour and other traditional ingredients. A thorough review of the state-of-the-art on pulse flour quality characterization reveals that research is required to elucidate the relationships between the micro- and nanoscale structures of these flours and their milling-dependent properties, such as hydration, starch and protein quality, components separation, and particle size distribution. With advances in synchrotron-enabled material characterization techniques, there exist a few options that have the potential to fill knowledge gaps. To this end, we conducted a comprehensive review of four high-resolution nondestructive techniques (i.e., scanning electron microscopy, synchrotron X-ray microtomography, synchrotron small-angle X-ray scattering, and Fourier-transformed infrared spectromicroscopy) and a comparison of their suitability for characterizing pulse flours. Our detailed synthesis of the literature concludes that a multimodal approach to fully characterize pulse flours will be vital to predicting their end-use suitability. A holistic characterization will help optimize and standardize the milling methods, pretreatments, and post-processing of pulse flours. Millers/processors will benefit by having a range of well-understood pulse flour fractions to incorporate into food formulations.

Cold and exogenous calcium alter Allium fistulosum cell wall pectin to depress intracellular freezing temperatures.

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca2+ is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 °C) and cold acclimated (2 weeks at 12/4 °C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhanced the temperature at which half the cells survived freezing injury by 8 °C (LT50 =-20 °C), and reduced tissue permeability by 70-fold compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca2+ in the cell wall, and a lower number of visible cell wall pores compared with that observed in cold acclimated plants. Using cryo-microscopy, we observed that 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells, but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate that the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells.

Transcriptomic analysis of differentially expressed genes in leaves and roots of two alfalfa (Medicago sativa L.) cultivars with different salt tolerance.

BACKGROUND: Alfalfa (Medicago sativa L.) production decreases under salt stress. Identification of genes associated with salt tolerance in alfalfa is essential for the development of molecular markers used for breeding and genetic improvement. RESULT: An RNA-Seq technique was applied to identify the differentially expressed genes (DEGs) associated with salt stress in two alfalfa cultivars: salt tolerant 'Halo' and salt intolerant 'Vernal'. Leaf and root tissues were sampled for RNA extraction at 0 h, 3 h, and 27 h under 12 dS m- 1 salt stress maintained by NaCl. The sequencing generated a total of 381 million clean sequence reads and 84.8% were mapped on to the alfalfa reference genome. A total of 237 DEGs were identified in leaves and 295 DEGs in roots of the two alfalfa cultivars. In leaf tissue, the two cultivars had a similar number of DEGs at 3 h and 27 h of salt stress, with 31 and 49 DEGs for 'Halo', 34 and 50 for 'Vernal', respectively. In root tissue, 'Halo' maintained 55 and 56 DEGs at 3 h and 27 h, respectively, while the number of DEGs decreased from 42 to 10 for 'Vernal'. This differential expression pattern highlights different genetic responses of the two cultivars to salt stress at different time points. Interestingly, 28 (leaf) and 31 (root) salt responsive candidate genes were highly expressed in 'Halo' compared to 'Vernal' under salt stress, of which 13 candidate genes were common for leaf and root tissues. About 60% of DEGs were assigned to known gene ontology (GO) categories. The genes were involved in transmembrane protein function, photosynthesis, carbohydrate metabolism, defense against oxidative damage, cell wall modification and protection against lipid peroxidation. Ion binding was found to be a key molecular activity for salt tolerance in alfalfa under salt stress. CONCLUSION: The identified DEGs are significant for understanding the genetic basis of salt tolerance in alfalfa. The generated genomic information is useful for molecular marker development for alfalfa genetic improvement for salt tolerance.

Specific Mycoparasite-Fusarium Graminearum Molecular Signatures in Germinating Seeds Disabled Fusarium Head Blight Pathogen's Infection.

Advances in Infrared (IR) spectroscopies have entered a new era of research with applications in phytobiome, plant microbiome and health. Fusarium graminearum 3-ADON is the most aggressive mycotoxigenic chemotype causing Fusarium head blight (FHB) in cereals; while Sphaerodes mycoparasitica is the specific Fusarium mycoparasite with biotrophic lifestyle discovered in cereal seeds and roots. Fourier transform infrared (FTIR) spectroscopy analyses depicted shifts in the spectral peaks related to mycoparasitism mainly within the region of proteins, lipids, also indicating a link between carbohydrates and protein regions, involving potential phenolic compounds. Especially, S. mycoparasitica contributes to significant changes in lipid region 3050-2800 cm-1, while in the protein region, an increasing trend was observed for the peaks 1655-1638 cm-1 (amide I) and 1549-1548 cm-1 (amide II) with changes in indicative protein secondary structures. Besides, the peak extending on the region 1520-1500 cm-1 insinuates a presence of aromatic compounds in presence of mycoparasite on the F. graminearum root sample. Monitoring shift in improved seed germination, fungus-fungus interface through scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), and FTIR molecular signatures combined with principal component analysis (PCA) proved useful tools to detect an early mycoparasitism as a vital asset of the preventive biocontrol strategy against plant pathogens.

Examining a synchrotron-based approach for in situ analyses of Al speciation in plant roots.

Aluminium (Al) K- and L-edge X-ray absorption near-edge structure (XANES) has been used to examine Al speciation in minerals but it remains unclear whether it is suitable for in situ analyses of Al speciation within plants. The XANES analyses for nine standard compounds and root tissues from soybean (Glycine max), buckwheat (Fagopyrum tataricum), and Arabidopsis (Arabidopsis thaliana) were conducted in situ. It was found that K-edge XANES is suitable for differentiating between tetrahedral coordination (peak of 1566 eV) and octahedral coordination (peak of 1568 to 1571 eV) Al, but not suitable for separating Al binding to some of the common physiologically relevant compounds in plant tissues. The Al L-edge XANES, which is more sensitive to changes in the chemical environment, was then examined. However, the poorer detection limit for analyses prevented differentiation of the Al forms in the plant tissues because of their comparatively low Al concentration. Where forms of Al differ markedly, K-edge analyses are likely to be of value for the examination of Al speciation in plant tissues. However, the apparent inability of Al K-edge XANES to differentiate between some of the physiologically relevant forms of Al may potentially limit its application within plant tissues, as does the poorer sensitivity at the L-edge.

Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism.

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.

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.

Genotypic and heat stress effects on leaf cuticles of field pea using ATR-FTIR spectroscopy.

MAIN CONCLUSION: ATR-FTIR spectroscopy in combination with uni- and multivariate analysis was used to quantify the spectral-chemical composition of the leaf cuticle of pea, investigating the effects of variety and heat stress. Field pea (Pisum sativum L.) is sensitive to heat stress and our goal was to improve canopy cooling and flower retention by investigating the protective role of lipid-related compounds in leaf cuticle, and to use results in the future to identify heat resistant genotypes. The objective was to use Attenuated Total Reflection (ATR)-Fourier Transform Infrared (FTIR) spectroscopy, a non-invasive technique, to investigate and quantify changes in adaxial cuticles of fresh leaves of pea varieties that were subjected to heat stress. Eleven varieties were grown under control (24/18 °C day/night) and heat stress conditions (35/18 °C day/night, for 5 days at the early flowering stage). These 11 had significant spectral differences in the integrated area of the main lipid region, CH2 region, CH3 peak, asymmetric and symmetric CH2 peaks, ester carbonyl peak, and the peak area ratio of CH2 to CH3 and ester carbonyl to CH2 asymmetric peak, indicating that cuticles had spectral-chemical diversity of waxes, cutin, and polysaccharides. Results indicated considerable diversity in spectral-chemical makeup of leaf cuticles within commercially available field pea varieties and they responded differently to high growth temperature, revealing their diverse potential to resist heat stress. The ATR-FTIR spectral technique can, therefore, be further used as a medium-throughput approach for rapid screening of superior cultivars for heat tolerance.

Pollen, ovules, and pollination in pea: Success, failure, and resilience in heat.

Field pea (Pisum sativum), a major grain legume crop, is autogamous and adapted to temperate climates. The objectives of this study were to investigate effects of high temperature stress on stamen chemical composition, anther dehiscence, pollen viability, pollen interactions with pistil and ovules, and ovule growth and viability. Two cultivars ("CDC Golden" and "CDC Sage") were exposed to 24/18°C (day/night) continually or to 35/18°C for 4 or 7 days. Heat stress altered stamen chemical composition, with lipid composition of "CDC Sage" being more stable compared with "CDC Golden." Heat stress reduced pollen viability and the proportion of ovules that received a pollen tube. After 4 days at 35°C, pollen viability in flower buds decreased in "CDC Golden," but not in "CDC Sage." After 7 days, partial to full failure of anthers to dehisce resulted in subnormal pollen loads on stigmas. Although growth (ovule size) of fertilized ovules was stimulated by 35°C, heat stress tended to decrease ovule viability. Pollen appears susceptible to stress, but not many grains are needed for successful fertilization. Ovule fertilization and embryos are less susceptible to heat, but further research is warranted to link the exact degree of resilience to stress intensity.

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.

Wheat flag leaf epicuticular wax morphology and composition in response to moderate drought stress are revealed by SEM, FTIR-ATR and synchrotron X-ray spectroscopy.

Wheat (Triticum aestivum L.) is the largest cereal crop grown in Western Canada where drought during late vegetative and seed filling stages affects plant development and yield. To identify new physiochemical markers associated with drought tolerance, epidermal characteristics of the flag leaf of two wheat cultivars with contrasting drought tolerance were investigated. The drought resistant 'Stettler' had a lower drought susceptibility index, greater harvest index and water-use efficiency than the susceptible 'Superb'. Furthermore, flag leaf width, relative water content and leaf roll were significantly greater in Stettler than in Superb at moderate drought stress (MdS). Visible differences in epicuticular wax density on the adaxial flag leaf surfaces and larger bulliform cells were identified in Stettler as opposed to Superb. Mid-infrared attenuated total internal reflectance spectra revealed that Stettler flag leaves had increased asymmetric and symmetric CH2 but reduced carbonyl esters on its adaxial leaf surface compared to Superb under MdS. X-ray fluorescence spectra revealed a significant increase in total flag leaf Zn concentrations in Stettler in response to MdS. Such information on the microstructural and chemical features of flag leaf may have potential as markers for drought tolerance and thereby accelerate the selection and release of more drought-resistant cultivars.

Evaluating Changes in Cell-Wall Components Associated with Clubroot Resistance Using Fourier Transform Infrared Spectroscopy and RT-PCR.

Clubroot disease is a serious threat to canola production in western Canada and many parts of the world. Rcr1 is a clubroot resistance (CR) gene identified recently and its molecular mechanisms in mediating CR have been studied using several omics approaches. The current study aimed to characterize the biochemical changes in the cell wall of canola roots connecting to key molecular mechanisms of this CR gene identified in prior studies using Fourier transform infrared (FTIR) spectroscopy. The expression of nine genes involved in phenylpropanoid metabolism was also studied using qPCR. Between susceptible (S) and resistance (R) samples, the most notable biochemical changes were related to an increased biosynthesis of lignin and phenolics. These results were supported by the transcription data on higher expression of BrPAL1. The up-regulation of PAL is indicative of an inducible defence response conferred by Rcr1; the activation of this basal defence gene via the phenylpropanoid pathway may contribute to clubroot resistance conferred by Rcr1. The data indicate that several cell-wall components, including lignin and pectin, may play a role in defence responses against clubroot. Principal components analysis of FTIR data separated non-inoculated samples from inoculated samples, but not so much between inoculated S and inoculated R samples. It is also shown that FTIR spectroscopy can be a useful tool in studying plant-pathogen interaction at cellular levels.

Synchrotron Radiation Sheds Fresh Light on Plant Research: The Use of Powerful Techniques to Probe Structure and Composition of Plants.

While synchrotron radiation is a powerful tool in material and biomedical sciences, it is still underutilized in plant research. This mini review attempts to introduce the potential of synchrotron-based spectroscopic and imaging methods and their applications to plant sciences. Synchrotron-based Fourier transform infrared spectroscopy, X-ray absorption and fluorescence techniques, and two- and three-dimensional imaging techniques are examined. We also discuss the limitations of synchrotron-based research in plant sciences, specifically the types of plant samples that can be used. Despite limitations, the unique features of synchrotron radiation such as high brightness, polarization and pulse properties offer great advantages over conventional spectroscopic and imaging tools and enable the correlation of the structure and chemical composition of plants with biochemical function. Modern detector technologies and experimental methodologies are thus enabling plant scientists to investigate aspects of plant sciences such as ultrafast kinetics of biochemical reactions, mineral uptake, transport and accumulation, and dynamics of cell wall structure and composition during environmental stress in unprecedented ways using synchrotron beamlines. The potential for the automation of some of these synchrotron technologies and their application to plant phenotyping is also discussed.

Synchrotron based phase contrast X-ray imaging combined with FTIR spectroscopy reveals structural and biomolecular differences in spikelets play a significant role in resistance to Fusarium in wheat.

BACKGROUND: Fusarium head blight (FHB), a scab principally caused by Fusarium graminearum Schw., is a serious disease of wheat. The purpose of this study is to evaluate the potential of combining synchrotron based phase contrast X-ray imaging (PCI) with Fourier Transform mid infrared (FTIR) spectroscopy to understand the mechanisms of resistance to FHB by resistant wheat cultivars. Our hypothesis is that structural and biochemical differences between resistant and susceptible cultivars play a significant role in developing resistance to FHB. RESULTS: Synchrotron based PCI images and FTIR absorption spectra (4000-800 cm(-1)) of the floret and rachis from Fusarium-damaged and undamaged spikes of the resistant cultivar 'Sumai3', tolerant cultivar 'FL62R1', and susceptible cultivar 'Muchmore' were collected and analyzed. The PCI images show significant differences between infected and non-infected florets and rachises of different wheat cultivars. However, no pronounced difference between non-inoculated resistant and susceptible cultivar in terms of floret structures could be determined due to the complexity of the internal structures. The FTIR spectra showed significant variability between infected and non-infected floret and rachis of the wheat cultivars. The changes in absorption wavenumbers following pathogenic infection were mostly in the spectral range from 1800-800 cm(-1). The Principal Component Analysis (PCA) was also used to determine the significant chemical changes inside floret and rachis when exposed to the FHB disease stress to understand the plant response mechanism. In the floret and rachis samples, PCA of FTIR spectra revealed differences in cell wall related polysaccharides. In the florets, absorption peaks for Amide I, cellulose, hemicellulose and pectin were affected by the pathogenic fungus. In the rachis of the wheat cultivars, PCA underlines significant changes in pectin, cellulose, and hemicellulose characteristic absorption spectra. Amide II and lignin absorption peaks, persistent in the rachis of Sumai3, together with increased peak shift at 1245 cm(-1) after infection with FHB may be a marker for stress response in which the cell wall compounds related to pathways for lignification are increased. CONCLUSIONS: Synchrotron based PCI combined with FTIR spectroscopy show promising results related to FHB in wheat. The combined technique is a powerful new tool for internal visualisation and biomolecular monitoring before and during plant-microbe interactions to understand both the differences between cultivars and their different responses to disease stress.

Seed set, pollen morphology and pollen surface composition response to heat stress in field pea.

Pea (Pisum sativum L.) is a major legume crop grown in a semi-arid climate in Western Canada, where heat stress affects pollination, seed set and yield. Seed set and pod growth characteristics, along with in vitro percentage pollen germination, pollen tube growth and pollen surface composition, were measured in two pea cultivars (CDC Golden and CDC Sage) subjected to five maximum temperature regimes ranging from 24 to 36 °C. Heat stress reduced percentage pollen germination, pollen tube length, pod length, seed number per pod, and the seed-ovule ratio. Percentage pollen germination of CDC Sage was greater than CDC Golden at 36 °C. No visible morphological differences in pollen grains or the pollen surface were observed between the heat and control-treated pea. However, pollen wall (intine) thickness increased due to heat stress. Mid-infrared attenuated total reflectance (MIR-ATR) spectra revealed that the chemical composition (lipid, proteins and carbohydrates) of each cultivar's pollen grains responded differently to heat stress. The lipid region of the pollen coat and exine of CDC Sage was more stable compared with CDC Golden at 36 °C. Secondary derivatives of ATR spectra indicated the presence of two lipid types, with different amounts present in pollen grains from each cultivar.

Soft X-ray spectromicroscopy study of mineral-organic matter associations in pasture soil clay fractions.

There is a growing acceptance that associations with soil minerals may be the most important overarching stabilization mechanism for soil organic matter. However, direct investigation of organo-mineral associations has been hampered by a lack of methods that can simultaneously characterize organic matter (OM) and soil minerals. In this study, STXM-NEXAFS spectroscopy at the C 1s, Ca 2p, Fe 2p, Al 1s, and Si 1s edges was used to investigate C associations with Ca, Fe, Al, and Si species in soil clay fractions from an upland pasture hillslope. Bulk techniques including C and N NEXAFS, Fe K-edge EXAFS spectroscopy, and XRD were applied to provide additional information. Results demonstrated that C was associated with Ca, Fe, Al, and Si with no separate phase in soil clay particles. In soil clay particles, the pervasive C forms were aromatic C, carboxyl C, and polysaccharides with the relative abundance of carboxyl C and polysaccharides varying spatially at the submicrometer scale. Only limited regions in the soil clay particles had aliphatic C. Good C-Ca spatial correlations were found for soil clay particles with no CaCO3, suggesting a strong role of Ca in organo-mineral assemblage formation. Fe EXAFS showed that about 50% of the total Fe in soils was contained in Fe oxides, whereas Fe-bearing aluminosilicates (vermiculite and Illite) accounted for another 50%. Fe oxides in the soil were mainly crystalline goethite and hematite, with lesser amounts of poorly crystalline ferrihydrite. XRD revealed that soil clay aluminosilicates were hydroxy-interlayered vermiculite, Illite, and kaolinite. C showed similar correlation with Fe to Al and Si, implying a similar association of Fe oxides and aluminosilicates with organic matter in organo-mineral associations. These direct microscopic determinations can help improve understanding of organo-mineral interactions in soils.

Synchrotron-based chemical nano-tomography of microbial cell-mineral aggregates in their natural, hydrated state.

Chemical nano-tomography of microbial cells in their natural, hydrated state provides direct evidence of metabolic and chemical processes. Cells of the nitrate-reducing Acidovorax sp. strain BoFeN1 were cultured in the presence of ferrous iron. Bacterial reduction of nitrate causes precipitation of Fe(III)-(oxyhydr)oxides in the periplasm and in direct vicinity of the cells. Nanoliter aliquots of cell-suspension were injected into custom-designed sample holders wherein polyimide membranes collapse around the cells by capillary forces. The immobilized, hydrated cells were analyzed by synchrotron-based scanning transmission X-ray microscopy in combination with angle-scan tomography. This approach provides three-dimensional (3D) maps of the chemical species in the sample by employing their intrinsic near-edge X-ray absorption properties. The cells were scanned through the focus of a monochromatic soft X-ray beam at different, chemically specific X-ray energies to acquire projection images of their corresponding X-ray absorbance. Based on these images, chemical composition maps were then calculated. Acquiring projections at different tilt angles allowed for 3D reconstruction of the chemical composition. Our approach allows for 3D chemical mapping of hydrated samples and thus provides direct evidence for the localization of metabolic and chemical processes in situ.