Mark David Fricker

Leaf venation architecture varies greatly among living and fossil plants. However, we still have a limited understanding of when, why and in which clades new architectures arose and how they impacted leaf functioning. Using data from 1,000 extant and extinct (fossil) plants, we reconstructed approximately 400 million years of venation evolution across clades and vein sizes. Overall, venation networks evolved from having fewer veins and less smooth loops to having more veins and smoother loops, but these changes only occurred in small and medium vein sizes. The diversity of architectural designs increased biphasically, first peaking in the Paleozoic, then decreasing during the Cretaceous, then increasing again in the Cenozoic, when recent angiosperm lineages initiated a second and ongoing phase of diversification. Vein evolution was not associated with temperature and CO₂ fluctuations but was associated with insect diversification. Our results highlight the complexity of the evolutionary trajectory and potential drivers of venation network architecture.

Summary: Variation in leaf venation network architecture may reflect trade‐offs among multiple functions including efficiency, resilience, support, cost, and resistance to drought and herbivory. However, our knowledge about architecture‐function trade‐offs is mostly based on studies examining a small number of functional axes, so we still lack a more integrative picture of multidimensional trade‐offs. Here, we measured architecture and functional traits on 122 ferns and angiosperms species to describe how trade‐offs vary across phylogenetic groups and vein spatial scales (small, medium, and large vein width) and determine whether architecture traits at each scale have independent or integrated effects on each function. We found that generalized architecture‐function trade‐offs are weak. Architecture strongly predicts leaf support and damage resistance axes but weakly predicts efficiency and resilience axes. Architecture traits at different spatial scales contribute to different functional axes, allowing plants to independently modulate different functions by varying network properties at each scale. This independence of vein architecture traits within and across spatial scales may enable evolution of multiple alternative leaf network designs with similar functioning.

Calcium (Ca ²⁺ ) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca ²⁺ -dynamics in fungi are poorly understood due to lack of effective real-time Ca ²⁺ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans , and used live-cell imaging to observe both dynamic Ca ²⁺ spiking and slower changes in non-spiking Ca ²⁺ -GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca ²⁺ -GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl _2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca ²⁺ -GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H ₂ O ₂ abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H ₂ O ₂ was dependent on Cap1, extracellular Ca ²⁺ , and calcineurin but not on its downstream target, Crz1. Ca ²⁺ -dynamics were not affected by H ₂ O ₂ in the hog1 Δ or yvc1 Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca ²⁺ -GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca ²⁺ -influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans . IMPORTANCE: Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans , for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

Calcium (Ca²⁺) is an important second messenger for activating stress response signalling and cell adaptation in eukaryotic cells yet intracellular Ca²⁺-dynamics in fungi is poorly understood due to lack of effective real-time Ca²⁺reporters. We engineered the GCaMP6f construct for use in the fungal pathogen,Candida albicans, and used live-cell imaging to observe dynamic Ca²⁺spiking as well as slower changes in ambient Ca²⁺-GCaMP levels elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca²⁺-GCaMP spiking was inhibited by 1 M NaCl but not by 0.66 M CaCl_2.Treatment with SDS caused a spike-burst, raised ambient Ca²⁺-GCaMP levels and significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H₂O₂abolished spiking and caused transient autofluorescence but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H₂O₂was dependent on Cap1, extracellular Ca²⁺and calcineurin, but not on its downstream target, Crz1. Ca²⁺-dynamics were not affected by H₂O₂in thehog1Δ oryvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca²⁺-GCaMP responses in individual cells has therefore revealed the dynamics of Ca²⁺-influx, signalling and homeostasis and their role in the temporal stress response signatures ofC. albicans.

Engineering of subcellular compartmentalisation is one of synthetic biology’s key challenges. Among different approaches, de novo construction of a synthetic compartment is the most coveted but also most difficult option. Restructuring the endoplasmic reticulum (ER), via the introduction of recombinant oligomerising ER-membrane resident proteins, is an alternative starting point for building a new compartment. The presence of such proteins leads to a massive expansion of the ER and the formation of organised smooth endoplasmic reticulum (OSER), a large membranous compartment. However, OSER is poorly characterised and our understanding of its effect on the underlying biology of the plant is limited. Here we characterise a range of OSER compartments and show how the structure of the inducing polyprotein constructs affect the final compartment morphology, with the cytosolic-facing antiparallel oligomerisation domain demonstrated to be an essential component to trigger OSER formation. We show that while OSER retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol. Using high-resolution quantitative image analysis, we also show that the presence of this large compartment does not disrupt the rest of the ER network. Moreover, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally. These properties collectively suggest that OSER could be developed as a plant synthetic biology tool for compartmentalisation, combining the benefits of several existing strategies. Only a single protein construct is necessary to induce its formation, and the compartment retains a delimiting membrane and a diffusional barrier to the rest of the cell.

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Colonization of terrestrial environments by filamentous fungi relies on their ability to form networks that can forage for and connect resource patches. Despite the importance of these networks, ecologists rarely consider network features as functional traits because their measurement and interpretation are conceptually and methodologically difficult. To address these challenges, we have developed a pipeline to translate images of fungal mycelia, from both micro- and macro-scales, to weighted network graphs that capture ecologically relevant fungal behaviour. We focus on four properties that we hypothesize determine how fungi forage for resources, specifically: connectivity; relative construction cost; transport efficiency; and robustness against attack by fungivores. Constrained ordination and Pareto front analysis of these traits revealed that foraging strategies can be distinguished predominantly along a gradient of connectivity for micro- and macro-scale mycelial networks that is reminiscent of the qualitative ‘phalanx’ and ‘guerilla’ descriptors previously proposed in the literature. At one extreme are species with many inter-connections that increase the paths for multidirectional transport and robustness to damage, but with a high construction cost; at the other extreme are species with an opposite phenotype. Thus, we propose this approach represents a significant advance in quantifying ecological strategies for fungi using network information.