Plants strategically allocate phosphorus (P) among leaf tissues to support physiological functions. However, there is limited understanding of contrasting leaf cellular P-allocation patterns and their physiological consequences. We quantified leaf P fractions, photosynthetic P-use efficiency (PPUE), and P-remobilisation efficiency (PRE) in five chickpea accessions exhibiting contrasting cellular P-allocation patterns. Leaf lifespan and concentrations of key phytohormones were also measured, including abscisic acid (ABA), salicylic acid (SA) and jasmonates. Cellular P-allocation patterns were more strongly associated with leaf lifespan than with PPUE or PRE. Accessions allocating a greater proportion of P to the mesophyll exhibited longer leaf lifespan, which was associated with lower jasmonate concentrations, rather than ABA. Surprisingly, greater P allocation to the epidermis was driven by a higher proportion of metabolite P. At the cellular level, we primarily observed a significant negative correlation between P and calcium concentrations in mesophyll cells, but not in the epidermis. Our findings provide the first evidence that leaf tissue-specific P-allocation patterns vary among chickpea accessions. Preferential allocation of P to photosynthetically active cells was associated with longer leaf lifespan. Differences in jasmonate concentrations accompanied these patterns. Collectively, these findings highlight new opportunities to enhance P-use efficiency in crop breeding programs.

Research at the interface between biology and materials science creates challenges for electron microscopists. Everything from the sample preparation to the choice of imaging and analytical techniques and the interpretation of the resulting data refuses to sit comfortably within the domain of one discipline or the other. Ultimately, these problems are best addressed by an interdisciplinary team that brings together expertise from both sides. While working in this environment can be challenging, with the need to learn new approaches, new language, and step outsides one's comfort zone, the benefits can be significant. Here we highlight those benefits using three examples of our own interdisciplinary collaborations (involving nanoparticles and other inorganic materials in plants, and nanostructures in butterflies) to encourage more electron microscopy researchers to reach out and embrace their colleagues on the other side of the interdisciplinary divide.

BACKGROUND AND AIM:Winter chilling has long been considered the primary driver of bud dormancy release in woody perennials. However, the cellular mechanisms underlying dormancy transitions remain poorly understood. This study aimed to investigate the relationship between dormancy depth, cell cycle activity, and cellular ultrastructure in grapevine buds across the dormancy cycle. METHODS:Bud dormancy progression in single node explants of Vitis vinifera cv. Cabernet Sauvignon was monitored from early autumn dormancy to the end of winter. Dormancy depth was quantified as the time to 50% bud burst under forcing conditions. Cell cycle status was assessed using quantitative flow cytometry, while the cellular ultrastructure of the shoot apical meristem was examined by transmission electron microscopy. KEY RESULTS:Dormancy depth declined dramatically from over 280 days in early autumn to ca 50 days in late autumn, despite negligible chilling exposure during this period. Following chilling exposure in winter, the depth of dormancy declined to ca 20 days. Strikingly, flow cytometry revealed that the majority of cells remained arrested in the G1 phase of mitosis throughout this period, regardless of dormancy depth, supported by ultrastructural analysis of the shoot apical meristem. Starch grains were abundant in both deeply dormant pre-winter buds (no chilling) and winter buds (with chilling), indicating that starch dynamics may be regulated independently of both dormancy status and chilling exposure. These findings demonstrate temporal uncoupling of dormancy release from cell cycle reactivation, challenging assumptions that dormancy release is directly linked to both chilling and cell cycle activation. CONCLUSION:Our results establish that in grapevine, the transition to growth competence occurs independently of detectable cell division and can precede significant winter chilling accumulation. This work provides new insights into the cellular basis of dormancy regulation in woody perennials and establishes a methodological framework for dissecting this relationship in other species.

The degree of plant silicification is classically proxied by organ-scale silicon concentration, [Si], which ignores where Si is allocated at the cellular scale. Bridging this scale gap is crucial to elucidate the physiological and ecological roles of silicification in high-Si taxa and its influence on biogeochemical cycles. We used high-resolution X-ray microtomography and cellular-scale element mapping to study silica allocation and silicification patterns in 15 species from the second most-abundant Si-accumulating family worldwide, namely Cyperaceae (sedges). Despite considerable variation in deposition sites and cellular [Si] between species and anatomical areas, we show that at least 78±17% of silica is localized in the epidermis, mostly as a silica layer in the outer tangential cell wall (OTW). Both the thickness and cellular-scale [Si] of this continuous layer, which is up to 18 µm thick, were highly correlated to organ-scale [Si] among species. Our findings demonstrate that bulk silica content is largely governed by deposition in the OTW in sedges, rather than by deposition in cell lumina. These findings open new perspectives for Si-related plant physiological and biogeochemical processes and pave the way for the use of X-ray microtomography not only to visualize, but also to quantify biogenic silica allocation in plants.

Introduction:Cryptosporidium hominis is the dominant Cryptosporidium species infecting humans, but most advances in developing robust in vitro culturing platforms for Cryptosporidium have utilised C. parvum. Consequently, there is relatively little available information specific to the biology and life cycle of C. hominis. The present study utilised a pumpless and tubeless gut-on-chip to generate a physiologically relevant in vitro environment by applying a constant fluid shear stress of 0.02 dyn cm-2 to HCT-8 cells. Methods:Gut-on-chips were fabricated using standard soft lithography. C. hominis oocysts isolated from human pathology samples were used to infect the human ileocecal colorectal adenocarcinoma (HCT-8) cell line under a constant fluid shear stress of 0.02 dyn cm-2. Parasite growth was assessed using a C. hominis-specific quantitative PCR, a Cryptosporidium genus-specific immunofluorescence assay, and scanning electron microscopy. Differences in the HCT-8 transcriptome with and without fluid shear stress, and the host-parasite interaction, were both assessed using bulk transcriptomics. Results:Transcriptomic analysis of the HCT-8 cell line cultured within the gut-on-chip demonstrated a metabolic shift towards oxidative phosphorylation when compared to the same cell line cultured under static conditions. Extended C. hominis (subtype IdA15G1) cultures were sustained for up to 10 days within the gut-on-chip as shown by a C. hominis-specific qPCR and a Cryptosporidium genus-specific immunofluorescence assay, which demonstrated ~30-fold amplification in the gut-on-chip over the duration of the experiment. Scanning electron microscopy of infected monolayers identified trophozoites, meronts, merozoites, macrogamonts, microgamonts, and possible gamont-like stages at 48 h post-infection. The potential role of gamonts in the Cryptosporidium life cycle remains unclear and warrants further investigation. Transcriptomes of HCT-8 cells infected with C hominis revealed upregulation of biological processes associated with cell cycle regulation and cell signalling in C. hominis-infected cells under fluid shear stress compared to static culture. Conclusions:These data demonstrate that bioengineered gut-on-chip models support extended C. hominis growth and can be used to interrogate responses of host cells to infection. Owing to its relative simplicity, the pumpless and tubeless gut-on-chip can be accessible to most laboratories with established HCT-8 infection models for Cryptosporidium culture.

Nature offers a remarkable diversity of nanomaterials that have extraordinary functional and structural properties. Intrinsic to nature is the impressive ability to form complex ordered nanomaterials via self-organization. One particularly intriguing nanostructure is the gyroid, a network-like structure exhibiting high symmetry and complex topology. Although its existence in cells and tissues across many biological kingdoms is well documented, how and why it forms remains elusive and uncovering these formation mechanisms will undoubtedly inform bioinspired designs. A beautiful example is the smooth single gyroid that is found in the wing scales of several butterflies, where it behaves as a photonic crystal generating a vibrant green color. Here, we report that the gyroid structures of the Emerald-patched Cattleheart, Parides sesostris, develop as woven fibrillar structures, in contrast to the commonly held assumption that they form as smooth constructs. Ultramicroscopy of pupal tissue reveals that the gyroid geometry consists of helical weavings of fibers, akin to hyperbolic line patterns decorating the gyroid. Interestingly, despite their fibrillar nature, electron diffraction reveals the absence of crystalline order within this material. Similar fibrillar structures are also observed in the mature wing scales of P. sesostris specimens with surgically altered pupal development, leading to a blue coloration. Our findings not only introduce a variation of the gyroid in biology but also have significant implications for our understanding of its formation in nature.

Objective: To investigate the effect of high intensity focused ultrasound (HIFU) exposure on enhancing the bonding of universal adhesives, in self-etch mode, to dentin. To achieve this aim, the effects of HIFU exposure on dentin micro-topography, structure, and nanoscale mechanical and chemical properties were investigated. In addition, resin-dentin interfacial morphology and micro-tensile bond strength (mu TBS) were evaluated. Methods: The effect of HIFU exposure at 5, 15, or 30W at varying time intervals (30-120 s) on the dentin surface properties were characterised by scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, and nano-indentation testing. The interfacial junctions between Scotchbond universal adhesive, in self-etch mode, and HIFU-treated dentin were characterised using confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Furthermore, mu TBS tests were conducted before and after thermocycling. Results: HIFU exposure at 30W for 120 s resulted in almost complete removal of the smear layer. HIFU treatment at 15W for 60 and 90 s, and at 30W for 60 s significantly increased dentin surface roughness. Raman analysis indicated progressive mineral removal at higher HIFU power levels. HIFU treatment at 5, 15, and 30W did not significantly affect dentin reduced elastic-modulus and nano-hardness. Following HIFU application at 30W for 120 s, CLSM examination indicated marked increase in adhesive penetration. HIFU exposure for 120 s at 30 W was effective in removing the overlying smear layer facilitating partial demineralisation of the underlying superficial dentin substrate. Whilst HIFU treatment did not improve the immediate mu TBS of Scotchbond universal adhesive in self-etch mode, 120 s HIFU treatment at 30W significantly improved the aged mu TBS values compared to the control. Significance: HIFU treatment of dentin surface at 30W for 120 s enhanced the structural integrity of resin-dentin interfacial junctions and the mu TBS, after thermocycling, of a mild universal adhesive applied in self-etch mode.

The physical property requirements for kaolinite suspensions to display time-dependent structural rebuilding or rejuvenation behavior are the same as required by smectite gels such as hectorite and Na-montmorillonite (NaMnt), a rare discovery linking the two types of clay. A holistic framework for predicting clay gel behavior based on the clay physical properties such as charge, crystal structure, and mineralogy, may emerge and was the ultimate goal of this research. A structural rejuvenation process during both ageing and stepdown shear rate modes was postulated to require that the silica and alumina faces of the kaolinite platelets be negatively charged to produce a strong electric double layer (EDL) repulsion in all face-face configurations. This is needed to slow down the (+)edge to (-)face bonding process to produce the time-dependent behavior. Currently, the unlike charge attraction between the silica and alumina faces makes the structural rebuilding process of a pre-sheared kaolinite suspension too fast to be observable. Two methods of making the alumina face negatively charged, i.e. the use of adsorbed P2O74- and high pH, did indeed produce the time-dependent behavior for both KGa-1b and KGa-2 suspensions, thus validating the proposed hypothesis. The KGa-1b with a lower content of octahedral positive layer charge required less P2O74- and a lower pH to achieve the desired outcome. Addition of 0.002 M NaCl to the high pH-treated KGa-2 suspension hastened the structural rejuvenation process and increased the aged gel strength. The oscillatory behavior in the stepdown shear stress at low pH was due to layer agglomerates formed by (+)face to (-)face attraction. Bulky layered agglomerates were reduced markedly by both the P2O74- and high pH treatments. The knowledge gained was applied successfully to make clay-rich iron ore tailings time-dependent in both the ageing and stepdown shear rate modes.

ABSTRACT Evaluating the cell cycle status during dormancy of multicellular organisms is problematic. This is particularly so for woody perennial buds, where dormant and quiescent states are diffuse, and the organ may remain visibly unchanged for six to nine months of the year. In this study, we investigate cell cycle status of dormant grapevine buds by measuring mitotic index using an optimised method developed for grapevine bud tissue. The experimental material showed a dynamic range in the depth of dormancy, declining from 200 days in March to less than 60 days in May and 30 days in August, measured as the time to reach 50% bud burst in forcing conditions. Despite these differences, flow cytometry analysis showed that most nuclei isolated from these buds were arrested at the G1 phase. Ultrastructure analysis of the cells in the region of the shoot apical meristem confirmed that the mitotic activities of buds remained low at all time points, together with the development of starch grains and the relative absence of organelle development. HIGHLIGHT The cell cycle and ultrastructure data suggest interesting evidence correspond to the growth resumption capacity of grapevine cv. Cabernet Sauvignon buds, i.e., absence of mitosis activities regardless of dormancy depth and starch accumulation irrespective of chilling accumulation.

An overlooked fraction of the terrestrial carbon (C) pool is that associated with biogenic silica deposited in plants (phytoliths), so-called PhytOC. This fraction is small compared with the main C pools, but is of interest because it could be a long-term C sink as phytoliths may protect organic C from mineralization. However, the topic is hotly contested and unclear due to both methodological and theoretical limitations. We aim to review this topic, with specific emphasis on: (i) the range of C concentrations associated with phytoliths; (ii) soil phytolith preservation and subsequent organic C mineralization; and (iii) global estimates of C sequestration within PhytOC. Recent work has suggested that [PhytOC] could be much greater than currently acknowledged, but also highly variable and dependent on cell silicification types. A short case study using cryo‐Scanning Electron Microscopy (cryo-SEM), X‐ray microanalysis (EDX), plus Focused Ion Beam (FIB) and Scanning Transmission Electron Microscopy (STEM) on the culms of a sedge (Schoenus caespititius) confirmed this thinking. Understanding of both phytolith and PhytOC fates in soil is poor. We suggest that phytolith residence time should be seen as a gradient. Such a continuum is explained by different phytolith sizes, types and chemistry, which will also have contrasting PhytOC. Our estimation of C sequestration as PhytOC each year (11–190 Tg C yr−1) represents between < 1

The sponge-like biomineralised calcite materials found in echinoderm skeletons are of interest in terms of both structure formation and biological function. Despite their crystalline atomic structure, they exhibit curved interfaces that have been related to known triply-periodic minimal surfaces. Here, we investigate the endoskeleton of the sea urchin Cidaris rugosa that has long been known to form a microstructure related to the Primitive surface. Using X-ray tomography, we find that the endoskeleton is organised as a composite material consisting of domains of bicontinuous microstructures with different structural properties. We describe, for the first time, the co-occurrence of ordered Primitive and Diamond structures and of a disordered structure within a single skeletal plate. We show that these structures can be distinguished by structural properties including solid volume fraction, trabeculae width, and to a lesser extent, interface area and mean curvature. In doing so, we present a robust method that extracts interface areas and curvature integrals from voxelized datasets using the Steiner polynomial for parallel body volumes. We discuss these very large scale bicontinuous structures in the context of their function, formation, and evolution.

In recent years, the detection of numerous negative correlations between silicon (Si) and carbon (C)-based compounds in plants has suggested trade-offs between different stress resistance and/or mechanical support strategies. However, nearly all studies have involved whole-leaf analysis, and it is unclear how the trade-off operates mechanistically, at the cellular level. Here we combined leaf trait measurements and microscopic analyses (electron microscopy with elemental X-ray mapping and X-ray microtomography) of 17 species from a high-Si family: Cyperaceae. Accumulation of Si was strongly negatively correlated with C-based compounds, particularly tannins. Our microscopical investigations showed that the accumulation of phenolics and deposition of silica were mutually exclusive in the outer epidermal cell walls. This trade-off was independent of that between the construction of tough, sclerenchyma-rich leaves and growth potential (the leaf economics spectrum). We also identified a strong negative correlation between Si and accumulation of epicuticular waxes. Previous whole leaf analyses were, in effect, hiding the locations of the trade-off between Si and C-based compounds in plants. The epidermal location of this trade-off and the specific involvement of tannins and waxes suggest the existence of different strategies to resist environmental stresses. Our study provides key insights into plant Si utilization and highlights the multidimensionality of plant stress resistance strategies.

Salinity tolerance requires coordinated responses encompassing salt exclusion in roots and tissue/cellular compartmentation of salt in leaves. We investigated the possible control points for salt ions transport in roots and tissue tolerance to Na+ and Cl- in leaves of two contrasting mungbean genotypes, salt-tolerant Jade AU and salt-sensitive BARI Mung-6, grown in nonsaline and saline (75 mM NaCl) soil. Cryo-SEM X-ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg, P, and S in various cell types in roots related to the development of apoplastic barriers, and in leaves related to photosynthetic performance. Jade AU exhibited superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis, and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell vacuoles compared to BARI Mung-6. Jade AU maintained higher [K] in root cells than BARI Mung-6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts and preferentially accumulated [K] in mesophyll cells than BARI Mung-6, resulting in higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in chloroplasts, and maintenance of high K in root and leaf mesophyll cells.

Biophotonic nanostructures in butterfly wing scales remain fascinating examples of biological functional materials, with intriguing open questions with regard to formation and evolutionary function. One particularly interesting butterfly species, Erora opisena (Lycaenidae: Theclinae), develops wing scales that contain three-dimensional photonic crystals that closely resemble a single gyroid geometry. Unlike most other gyroid-forming butterflies, E. opisena develops discrete gyroid crystallites with a pronounced size gradient hinting at a developmental sequence frozen in time. Here, we present a novel application of a hyperspectral (wavelength-resolved) microscopy technique to investigate the ultrastructural organization of these gyroid crystallites in dry, adult wing scales. We show that reflectance corresponds to crystallite size, where larger crystallites reflect green wavelengths more intensely; this relationship could be used to infer size from the optical signal. We further successfully resolve the red-shifted reflectance signal from wing scales immersed in refractive index liquids with varying refractive index, including values similar to water or cytosol. Such photonic crystals with lower refractive index contrast may be similar to the hypothesized nanostructural forms in the developing butterfly scales. The ability to resolve these fainter signals hints at the potential of this facile light microscopy method for in vivo analysis of nanostructure formation in developing butterflies.

Salt strengthened the bond formed and quickened the bonding process of 3-5 wt. % hectorite gels during the structural rejuvenation process. This even occurred at 0.002M KCl. Microstructure showed exfoliated, flexible platelet bonding in (+)edge-(-)face configurations. The display of prominent aging time-dependent behavior is due to the structural rejuvenation process being controlled by the electric double layer (EDL) repulsive force. Salt increased the lower energy paths to bonding in the (+)edge-(-)face configurations and weakened the EDL force to form stronger bonds. The Leong model time constant data supported the faster bonding process. In shear, the gels with a weakened EDL repulsive force caused by 0.01 and 0.1M KCl treatment were unable to display EDL force-control time-dependent behavior in the stepdown shear stress response. This situation was remedied by increasing the negative charge density of platelets with adsorbed P2O74-. The amount of P2O74- needed was higher at 0.1M KCl. (c) 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).https://doi.org/10.1063/5.0241130

Background and aims Proteaceae are a prominent plant family in south-western Australia. Most Proteaceae are ‘calcifuge’, occurring exclusively on old phosphorus (P)-impoverished acidic soils, with a few ‘soil-indifferent’ species also found on young P-richer calcareous soils. Calcium (Ca)-enhanced P toxicity explains the calcifuge habit of Proteaceae. However, previous research has so far been focused exclusively on the roles of Ca and P in determining Proteaceae distribution, and consequently there is little knowledge on how other soil-based strategies influence this distribution. We aimed to study the effects of young calcareous soils on four soil-grown Proteaceae and assess differences between calcifuge and soil-indifferent Proteaceae to better understand their natural distribution. Methods Two calcifuge and two soil-indifferent Proteaceae from south-western Australia were grown in six contrasting soils, including young calcareous, and old acidic soils. Results When grown in calcareous soils all species showed root growth inhibition, micronutrient deficiency, Ca-enhanced P toxicity, and negative impacts on physiology. Calcifuge species were more sensitive to calcareous soils than soil-indifferent ones, although this varied between genera. Soil-indifferent species tended to produce more cluster roots, release more carboxylates per root mass, and allocate less Ca to their leaves, compared with calcifuges; they also had smaller seeds and were less sensitive to Ca-enhanced P toxicity. Conclusion We surmise that a combination of these traits allows soil-indifferent species to tolerate calcareous soils. This study provides insight into how Proteaceae respond to young calcareous soils and how this influences their distribution.

Behaviours such as chemotaxis can facilitate metabolic exchanges between phytoplankton and heterotrophic bacteria, which ultimately regulate oceanic productivity and biogeochemistry. However, numerically dominant picophytoplankton have been considered too small to be detected by chemotactic bacteria, implying that cell-cell interactions might not be possible between some of the most abundant organisms in the ocean. Here we examined how bacterial behaviour influences metabolic exchanges at the single-cell level between the ubiquitous picophytoplankton Synechococcus and the heterotrophic bacterium Marinobacter adhaerens, using bacterial mutants deficient in motility and chemotaxis. Stable-isotope tracking revealed that chemotaxis increased nitrogen and carbon uptake of both partners by up to 4.4-fold. A mathematical model following thousands of cells confirmed that short periods of exposure to small but nutrient-rich microenvironments surrounding Synechococcus cells provide a considerable competitive advantage to chemotactic bacteria. These findings reveal that transient interactions mediated by chemotaxis can underpin metabolic relationships among the ocean's most abundant microorganisms.

Composite NaMnt (SWy-2)-kaolin (KGa-2) gels at more than 10 wt% solids with the NaMnt fraction = 20% formed an open cellular microstructure. The flexible, nanosized NaMnt platelets formed the continuous structure with kaolin particles dispersed within it bonding with the NaMnt platelets. The morphology adopted by the single layer NaMnt platelets is varied and complex producing a wide range of unique particle interaction configurations. Many acquired a curved shape surface with curled edges. Some of the platelets developed elongated string-like features made by the curling of edges and may have 3-5 of these strings for bonding. Kaolin particles are rigid and multilayered, and most have a platelet morphology. A common NaMnt-kaolin bonding configuration observed was the face at the curled-edge of NaMnt platelet resting on the face of the kaolin particle. A 15.5 wt% gel with a 1:2 mass ratio of NaMnt to kaolin was demonstrated to display both thixotropic and rheopectic behavior. A stepdown shear rate method was used to evaluate the structural development kinetics at 10 s(-1) of this gel commencing with the structural state sheared to equilibrium at 1000 s(-1). The Leong model described this kinetics well and found that the structural development characteristic time to be very fast, in seconds. The ageing behavior was also evaluated without the imposition of a constant shear. The yield stress increase continued for weeks and months for the 2:1 kaolin to NaMnt composite gels at 15.5 and 10.7 wt% solids. The characteristic time of the structural development process was much longer, in days. Upon reducing the NaMnt content from 33.3% to 20%, the 10.8% wt% gel became weakly time-dependent with a marginal yield stress. A continuous structure cannot be formed immediately upon the cessation of shear due to an inadequate NaMnt platelet concentration.

Although significant intraspecific variation in photosynthetic phosphorus (P) use efficiency (PPUE) has been shown in numerous species, we still know little about the biochemical basis for differences in PPUE among genotypes within a species. Here, we grew two high PPUE and two low PPUE chickpea (Cicer arietinum) genotypes with low P supply in a glasshouse to compare their photosynthesis-related traits, total foliar P concentration ([P]) and chemical P fractions (i.e. inorganic P (Pi), metabolite P, lipid P, nucleic acid P, and residual P). Foliar cell-specific nutrient concentrations including P were characterized using elemental X-ray microanalysis. Genotypes with high PPUE showed lower total foliar [P] without slower photosynthetic rates. No consistent differences in cellular [P] between the epidermis and mesophyll cells occurred across the four genotypes. In contrast, high PPUE was associated with lower allocation to Pi and metabolite P, with PPUE being negatively correlated with the percentage of these two fractions. Furthermore, a lower allocation to Pi and metabolite P was correlated with a greater allocation to nucleic acid P, but not to lipid P. Collectively, our results suggest that a different allocation to foliar P fractions, rather than preferential P allocation to specific leaf tissues, underlies the contrasting PPUE among chickpea genotypes.

To study the time-dependent properties of clay gels in the structural rejuvenation mode, a combination of the stepdown shear rate method and an ageing test is advocated. In this mode, the structural development process at two different initial equilibrium states during the low stepdown shear rate and the rest period of the ageing test were followed. The roles played by the electric double layer (EDL) repulsive force in this structural development process with and without shear, for platelet sodium montmorillonite or NaMnt, hectorite and Laponite gels, and fibrous sepiolite gels were assessed. The response of the stepdown shear stress and ageing yield stress reflects the action of these forces on the structural rejuvenation kinetics and the type of structure formed. The platelet clay gels with their strong EDL repulsive particle interaction commenced the structural development process immediately and formed an open network structure. This process is short in shear and takes years when without. The fibrous sepiolite gel with its weak EDL repulsive interaction did not display structural rejuvenation in shear and only showed a significant yield stress increase after the first few hours of ageing. An additional issue concerning the stability of the isotropic liquid (IL) phase state of dilute Laponite dispersions was addressed. These dispersions aged for >9 years, displaying that yield stress and network structure confirmed that the IL phase is unstable. A low energy path available for Laponite particles to aggregate was discussed. A new method for determining the flocculate phase state was also presented.