Starch depletion in the xylem and phloem ray parenchyma of grapevine stems under drought.

While nonstructural carbohydrate (NSC) storage can support long-lived woody plants during abiotic stress, the timing and extent of their use are less understood, as are the thresholds for cell mortality as NSCs and water supplies are consumed. Here, we combine physiological and imaging tools to study the response of Vitis riparia to a 6-week experimental drought. We focused on the spatial and temporal dynamics of starch consumption and cell viability in the xylem and phloem of the stem. Starch dynamics were further corroborated with enzymatic starch digestion and X-ray microcomputed tomography imaging. Starch depletion in the stems of droughted plants was detected after 2 weeks and continued over time. We observed distinct differences in starch content and cell viability in the xylem and phloem. By the end of the drought, nearly all the starch was consumed in the phloem ray parenchyma (98 % decrease), and there were almost no metabolically active cells in the phloem. In contrast, less starch was consumed in the xylem ray parenchyma (30 % decrease), and metabolically active cells remained in the ray and vessel-associated parenchyma in the xylem. Our data suggest that the higher proportion of living cells in the phloem and cambium, combined with smaller potential NSC storage area, rapidly depleted starch, which led to cell death. In contrast, the larger cross-sectional area of the xylem ray parenchyma with higher NSC storage and lower metabolically active cell populations depleted starch at a slower pace. Why NSC source-sink relationships between xylem and phloem do not allow for a more uniform depletion of starch in ray parenchyma over time is unclear. Our data help to pinpoint the proximate and ultimate causes of plant death during prolonged drought exposure and highlight the need to consider the influence of within-organ starch dynamics and cell mortality on abiotic stress response.

Anatomy of an agricultural antagonist: Feeding complex structure and function of three xylem sap-feeding insects illuminated with synchrotron-based 3D imaging.

Many insects feed on xylem or phloem sap of vascular plants. Although physical damage to the plant is minimal, the process of insect feeding can transmit lethal viruses and bacterial pathogens. Disparities between insect-mediated pathogen transmission efficiency have been identified among xylem sap-feeding insects; however, the mechanistic drivers of these trends are unclear. Identifying and understanding the structural factors and associated integrated functional components that may ultimately determine these disparities are critical for managing plant diseases. Here, we applied synchrotron-based X-ray microcomputed tomography to digitally reconstruct the morphology of three xylem sap-feeding insect vectors of plant pathogens: Graphocephala atropunctata (blue-green sharpshooter; Hemiptera, Cicadellidae) and Homalodisca vitripennis (glassy-winged sharpshooter; Hemiptera, Cicadellidae), and the spittlebug Philaenus spumarius (meadow spittlebug; Hemiptera, Aphrophoridae). The application of this technique revealed previously undescribed anatomical features of these organisms, such as key components of the salivary complex. The visualization of the 3D structure of the precibarial valve led to new insights into the mechanism of how this structure functions. Morphological disparities with functional implications between taxa were highlighted as well, including the morphology and volume of the cibarial dilator musculature responsible for extracting xylem sap, which has implications for force application capabilities. These morphological insights will be used to target analyses illuminating functional differences in feeding behavior.

Back to life: Techniques for developing high-quality 3D reconstructions of plants and animals from digitized specimens.

Expanded use of 3D imaging in organismal biology and paleontology has substantially enhanced the ability to visualize and analyze specimens. These techniques have improved our understanding of the anatomy of many taxa, and the integration of downstream computational tools applied to 3D datasets have broadened the range of analyses that can be performed (e.g., finite element analyses, geometric morphometrics, biomechanical modeling, physical modeling using 3D printing). However, morphological analyses inevitably present challenges, particularly in fossil taxa where taphonomic or preservational artifacts distort and reduce the fidelity of the original morphology through shearing, compression, and disarticulation, for example. Here, we present a compilation of techniques to build high-quality 3D digital models of extant and fossil taxa from 3D imaging data using freely available software for students and educators. Our case studies and associated step-by-step supplementary tutorials present instructions for working with reconstructions of plants and animals to directly address and resolve common issues with 3D imaging data. The strategies demonstrated here optimize scientific accuracy and computational efficiency and can be applied to a broad range of taxa.

Localized growth and remodelling drives spongy mesophyll morphogenesis.

The spongy mesophyll is a complex, porous tissue found in plant leaves that enables carbon capture and provides mechanical stability. Unlike many other biological tissues, which remain confluent throughout development, the spongy mesophyll must develop from an initially confluent tissue into a tortuous network of cells with a large proportion of intercellular airspace. How the airspace in the spongy mesophyll develops while the tissue remains mechanically stable is unknown. Here, we use computer simulations of deformable polygons to develop a purely mechanical model for the development of the spongy mesophyll tissue. By stipulating that cell wall growth and remodelling occurs only near void space, our computational model is able to recapitulate spongy mesophyll development observed in Arabidopsis thaliana leaves. We find that robust generation of pore space in the spongy mesophyll requires a balance of cell growth, adhesion, stiffness and tissue pressure to ensure cell networks become porous yet maintain mechanical stability. The success of this mechanical model of morphogenesis suggests that simple physical principles can coordinate and drive the development of complex plant tissues like the spongy mesophyll.

Hydraulic failure as a primary driver of xylem network evolution in early vascular plants.

The earliest vascular plants had stems with a central cylindrical strand of water-conducting xylem, which rapidly diversified into more complex shapes. This diversification is understood to coincide with increases in plant body size and branching; however, no selection pressure favoring xylem strand-shape complexity is known. We show that incremental changes in xylem network organization that diverge from the cylindrical ancestral form lead to progressively greater drought resistance by reducing the risk of hydraulic failure. As xylem strand complexity increases, independent pathways for embolism spread become fewer and increasingly concentrated in more centrally located conduits, thus limiting the systemic spread of embolism during drought. Selection by drought may thus explain observed trajectories of xylem strand evolution in the fossil record and the diversity of extant forms.

Using X-ray Micro-Computed Tomography to Three-Dimensionally Visualize the Foregut of the Glassy-Winged Sharpshooter (Homalodisca vitripennis).

Dissecting the heads of small insects belonging to the order Hemiptera for detailed anatomical investigation with light or scanning electron microscopy is difficult, time-consuming, and destructive, often resulting in sample preparation artifacts. Nevertheless, the structural details of these insects often hold critical information regarding their interactions with bacterial pathogens. For example, the glassy-winged sharpshooter (GWSS) is an efficient vector of the bacterium Xylella fastidiosa, the causal agent of Pierce's disease in grape vines, but the foregut of this species is exceptionally difficult to dissect. Here, we describe a simple, non-destructive method to investigate the structure of the anterior gut of GWSS using high-resolution micro-computed X-ray tomography (microCT). The use of microCT eliminates the need for destructive dissection and reveals the morphology of small insects in three dimensions, allowing the user to virtually dissect the sample. The use of microCT imaging is a promising and powerful tool in the entomological sciences for studying the structures of vector insects, especially for difficult-to-dissect regions such as the foregut.

Pathogen-induced hydraulic decline limits photosynthesis and starch storage in grapevines (Vitis sp.).

Xylella fastidiosa (Xf) is the bacterial pathogen responsible for Pierce's Disease (PD) in grapevine (Vitis vinifera L.) and numerous diseases in agriculturally and ecologically important species. Current theory suggests that localized inoculations via insect feeding lead to bacterial spread through the xylem, reducing water transport capacity, leading to declines in productivity, and ultimately death. Yet, the underlying mechanisms of Xf-induced mortality are not fully understood. In this study, we documented the development of PD symptoms over 12-13 weeks postinoculation. Subsequently assessed photosynthetic capacity, starch storage, and stem hydraulics in four grapevine genotypes (two PD-resistant and two PD-susceptible), comparing those physiological changes to control plants. PD-susceptible genotypes showed a coordinated decline in photosynthesis, starch storage, and stem hydraulics, whereas Xf-inoculation led to no change in starch and stem hydraulics in the PD-resistant genotypes. Together these data support the idea of a link between loss of hydraulic conductivity due to tylosis production with a downstream photosynthetic decline and starch depletion in the PD-susceptible genotypes. Our data support the theory that hydraulic failure and carbon starvation underlie plant mortality resulting from PD.

Desiccation of the leaf mesophyll and its implications for CO2 diffusion and light processing.

Leaves balance CO2 and radiative absorption while maintaining water transport to maximise photosynthesis. Related species with contrasting leaf anatomy can provide insights into inherent and stress-induced links between structure and function for commonly measured leaf traits for important crops. We used two walnut species with contrasting mesophyll anatomy to evaluate these integrated exchange processes under non-stressed and drought conditions using a combination of light microscopy, X-ray microCT, gas exchange, hydraulic conductance, and chlorophyll distribution profiles through leaves. Juglans regia had thicker palisade mesophyll, higher fluorescence in the palisade, and greater low-mesophyll porosity that were associated with greater gas-phase diffusion (gIAS ), stomatal and mesophyll (gm ) conductances and carboxylation capacity. More and highly-packed mesophyll cells and bundle sheath extensions (BSEs) in Juglans microcarpa led to higher fluorescence in the spongy and in proximity to the BSEs. Both species exhibited drought-induced reductions in mesophyll cell volume, yet the associated increases in porosity and gIAS were obscured by declines in biochemical activity that decreased gm . Inherent differences in leaf anatomy between the species were linked to differences in gas exchange, light absorption and photosynthetic capacity, and drought-induced changes in leaf structure impacted performance via imposing species-specific limitations to light absorption, gas exchange and hydraulics.

Structural organization of the spongy mesophyll.

Many plant leaves have two layers of photosynthetic tissue: the palisade and spongy mesophyll. Whereas palisade mesophyll consists of tightly packed columnar cells, the structure of spongy mesophyll is not well characterized and often treated as a random assemblage of irregularly shaped cells. Using micro-computed tomography imaging, topological analysis, and a comparative physiological framework, we examined the structure of the spongy mesophyll in 40 species from 30 genera with laminar leaves and reticulate venation. A spectrum of spongy mesophyll diversity encompassed two dominant phenotypes: first, an ordered, honeycomblike tissue structure that emerged from the spatial coordination of multilobed cells, conforming to the physical principles of Euler's law; and second, a less-ordered, isotropic network of cells. Phenotypic variation was associated with transitions in cell size, cell packing density, mesophyll surface-area-to-volume ratio, vein density, and maximum photosynthetic rate. These results show that simple principles may govern the organization and scaling of the spongy mesophyll in many plants and demonstrate the presence of structural patterns associated with leaf function. This improved understanding of mesophyll anatomy provides new opportunities for spatially explicit analyses of leaf development, physiology, and biomechanics.

Desiccation and rehydration dynamics in the epiphytic resurrection fern Pleopeltis polypodioides.

The epiphytic resurrection-or desiccation-tolerant (DT)-fern Pleopeltis polypodioides can survive extreme desiccation and recover physiological activity within hours of rehydration. Yet, how epiphytic DT ferns coordinate between deterioration and recovery of their hydraulic and photosynthetic systems remains poorly understood. We examined the functional status of the leaf vascular system, chlorophyll fluorescence, and photosynthetic rate during desiccation and rehydration of P. polypodioides. Xylem tracheids in the stipe embolized within 3-4 h during dehydration. When the leaf and rhizome received water, tracheids refilled after ∼24 h, which occurred along with dramatic structural changes in the stele. Photosynthetic rate and chlorophyll fluorescence recovered to predesiccation values within 12 h of rehydration, regardless of whether fronds were connected to their rhizome. Our data show that the epiphytic DT fern P. polypodioides can utilize foliar water uptake to rehydrate the leaf mesophyll and recover photosynthesis despite a broken hydraulic connection to the rhizome.

Ecologically driven selection of nonstructural carbohydrate storage in oak trees.

Leaf habit is a major axis of plant diversity that has consequences for carbon balance since the leaf is the primary site of photosynthesis. Nonstructural carbohydrates (NSCs) produced by photosynthesis can be allocated to storage and serve as a resiliency mechanism to future abiotic and biotic stress. However, how leaf habit affects NSC storage in an evolutionary context has not been shown. Using a comparative physiological framework and an analysis of evolutionary model fitting, we examined if variation in NSC storage is explained by leaf habit. We measured sugar and starch concentrations in 51 oak species (Quercus spp.) growing in a common garden and representing multiple evolutions of three different leaf habits (deciduous, brevideciduous and evergreen). The best fitting evolutionary models indicated that deciduous oak species are evolving towards higher NSC concentrations than their brevideciduous and evergreen relatives. Notably, this was observed for starch (the primary storage molecule) in the stem (a long-term C storage organ). Overall, our work provides insight into the evolutionary drivers of NSC storage and suggests that a deciduous strategy may confer an advantage against stress associated with a changing world. Future work should examine additional clades to further corroborate this idea.

Linking xylem network failure with leaf tissue death.

Global warming is expected to dramatically accelerate forest mortality as temperature and drought intensity increase. Predicting the magnitude of this impact urgently requires an understanding of the process connecting atmospheric drying to plant tissue damage. Recent episodes of forest mortality worldwide have been widely attributed to dry conditions causing acute damage to plant vascular systems. Under this scenario vascular embolisms produced by water stress are thought to cause plant death, yet this hypothetical trajectory has never been empirically demonstrated. Here we provide foundational evidence connecting failure in the vascular network of leaves with tissue damage caused during water stress. We observe a catastrophic sequence initiated by water column breakage under tension in leaf veins which severs local leaf tissue water supply, immediately causing acute cellular dehydration and irreversible damage. By highlighting the primacy of vascular network failure in the death of leaves exposed to drought or evaporative stress our results provide a strong mechanistic foundation upon which models of plant damage in response to dehydration can be confidently structured.

Maximum CO2 diffusion inside leaves is limited by the scaling of cell size and genome size.

Maintaining high rates of photosynthesis in leaves requires efficient movement of CO2 from the atmosphere to the mesophyll cells inside the leaf where CO2 is converted into sugar. CO2 diffusion inside the leaf depends directly on the structure of the mesophyll cells and their surrounding airspace, which have been difficult to characterize because of their inherently three-dimensional organization. Yet faster CO2 diffusion inside the leaf was probably critical in elevating rates of photosynthesis that occurred among angiosperm lineages. Here we characterize the three-dimensional surface area of the leaf mesophyll across vascular plants. We show that genome size determines the sizes and packing densities of cells in all leaf tissues and that smaller cells enable more mesophyll surface area to be packed into the leaf volume, facilitating higher CO2 diffusion. Measurements and modelling revealed that the spongy mesophyll layer better facilitates gaseous phase diffusion while the palisade mesophyll layer better facilitates liquid-phase diffusion. Our results demonstrate that genome downsizing among the angiosperms was critical to restructuring the entire pathway of CO2 diffusion into and through the leaf, maintaining high rates of CO2 supply to the leaf mesophyll despite declining atmospheric CO2 levels during the Cretaceous.

Diversification, disparification and hybridization in the desert shrubs Encelia.

There are multiple hypotheses for the spectacular plant diversity found in deserts. We explore how different factors, including the roles of ecological opportunity and selection, promote diversification and disparification in Encelia, a lineage of woody plants in the deserts of the Americas. Using a nearly complete species-level phylogeny based on double-digest restriction-aided sequencing along with a broad set of phenotypic traits, we estimate divergence times and diversification rates, identify instances of hybridization, quantify trait disparity and assess phenotypic divergence across environmental gradients. We show that Encelia originated and diversified recently (mid-Pleistocene) and rapidly, with rates comparable to notable adaptive radiations in plants. Encelia probably originated in the hot deserts of North America, with subsequent diversification across steep environmental gradients. We uncover multiple instances of gene flow between species. The radiation of Encelia is characterized by fast rates of phenotypic evolution, trait lability and extreme disparity across environments and between species pairs with overlapping geographic ranges. Encelia exemplifies how interspecific gene flow in combination with high trait lability can enable exceptionally fast diversification and disparification across steep environmental gradients.

Differences in grapevine rootstock sensitivity and recovery from drought are linked to fine root cortical lacunae and root tip function.

Structural changes during severe drought stress greatly modify the hydraulic properties of fine roots. Yet, the physiological basis behind the restoration of fine root water uptake capacity during water recovery remains unknown. Using neutron radiography (NR), X-ray micro-computed tomography (micro-CT), fluorescence microscopy, and fine root hydraulic conductivity measurements (Lpr ), we examined how drought-induced changes in anatomy and hydraulic properties of contrasting grapevine rootstocks are coupled with fine root growth dynamics during drought and return of soil moisture. Lacunae formation in drought-stressed fine roots was associated with a significant decrease in fine root Lpr for both rootstocks. However, lacunae formation occurred under milder stress in the drought-resistant rootstock, 110R. Suberin was deposited at an earlier developmental stage in fine roots of 101-14Mgt (i.e. drought susceptible), probably limiting cortical lacunae formation during mild stress. During recovery, we found that only 110R fine roots showed rapid re-establishment of elongation and water uptake capacity and we found that soil water status surrounding root tips differed between rootstocks as imaged with NR. These data suggest that drought resistance in grapevine rootstocks is associated with rapid re-establishment of growth and Lpr near the root tip upon re-watering by limiting competing sites along the root cylinder.

Coordinated decline of leaf hydraulic and stomatal conductances under drought is not linked to leaf xylem embolism for different grapevine cultivars.

Drought decreases water transport capacity of leaves and limits gas exchange, which involves reduced leaf leaf hydraulic conductance (Kleaf) in both the xylem and outside-xylem pathways. Some literature suggests that grapevines are hyper-susceptible to drought-induced xylem embolism. We combined Kleaf and gas exchange measurements, micro-computed tomography of intact leaves, and spatially explicit modeling of the outside-xylem pathways to evaluate the role of vein embolism and Kleaf in the responses of two different grapevine cultivars to drought. Cabernet Sauvignon and Chardonnay exhibited similar vulnerabilities of Kleaf and gs to dehydration, decreasing substantially prior to leaf xylem embolism. Kleaf and gs decreased by 80% for both cultivars by Ψ leaf approximately -0.7 MPa and -1.2 MPa, respectively, while leaf xylem embolism initiated around Ψ leaf = -1.25 MPa in the midribs and little to no embolism was detected in minor veins even under severe dehydration for both cultivars. Modeling results indicated that reduced membrane permeability associated with a Casparian-like band in the leaf vein bundle sheath would explain declines in Kleaf of both cultivars. We conclude that during moderate water stress, changes in the outside-xylem pathways, rather than xylem embolism, are responsible for reduced Kleaf and gs. Understanding this mechanism could help to ensure adequate carbon capture and crop performance under drought.

Seasonal coordination of leaf hydraulics and gas exchange in a wintergreen fern.

Wintergreen fern Polystichum acrostichoides has fronds that are photosynthetically active year-round, despite diurnal and seasonal changes in soil moisture, air temperature and light availability. This species can fix much of its annual carbon during periods when the deciduous canopy is open. Yet, remaining photosynthetically active year-round requires the maintenance of photosynthetic and hydraulic systems that are vulnerable to freeze-thaw cycles. We aimed to determine the anatomical and physiological strategies P. acrostichoides uses to maintain positive carbon gain, and the coordination between the hydraulic and photosynthetic systems. We found that the first night below 0 °C led to 25 % loss of conductivity (PLC) in stipes, suggesting that winter-induced embolism occurred. Maximum photosynthetic rate and chlorophyll fluorescence declined during winter but recovered by spring, despite PLC remaining high; the remaining hydraulic capacity was sufficient to supply the leaves with water. The onset of colder temperatures coincided with the development of a necrotic hinge zone at the stipe base, allowing fronds to overwinter lying prostrate and maintain a favourable leaf temperature. Our conductivity data show that the hinge zone did not affect leaf hydraulics because of the flexibility of the vasculature. Collectively, these strategies help P. acrostichoides to survive in northeastern forests.

Natural selection maintains species despite frequent hybridization in the desert shrub Encelia.

Natural selection is an important driver of genetic and phenotypic differentiation between species. For species in which potential gene flow is high but realized gene flow is low, adaptation via natural selection may be a particularly important force maintaining species. For a recent radiation of New World desert shrubs (Encelia: Asteraceae), we use fine-scale geographic sampling and population genomics to determine patterns of gene flow across two hybrid zones formed between two independent pairs of species with parapatric distributions. After finding evidence for extremely strong selection at both hybrid zones, we use a combination of field experiments, high-resolution imaging, and physiological measurements to determine the ecological basis for selection at one of the hybrid zones. Our results identify multiple ecological mechanisms of selection (drought, salinity, herbivory, and burial) that together are sufficient to maintain species boundaries despite high rates of hybridization. Given that multiple pairs of Encelia species hybridize at ecologically divergent parapatric boundaries, such mechanisms may maintain species boundaries throughout Encelia.

Digitally deconstructing leaves in 3D using X-ray microcomputed tomography and machine learning.

PREMISE: X-ray microcomputed tomography (microCT) can be used to measure 3D leaf internal anatomy, providing a holistic view of tissue organization. Previously, the substantial time needed for segmenting multiple tissues limited this technique to small data sets, restricting its utility for phenotyping experiments and limiting our confidence in the inferences of these studies due to low replication numbers. METHODS AND RESULTS: We present a Python codebase for random forest machine learning segmentation and 3D leaf anatomical trait quantification that dramatically reduces the time required to process single-leaf microCT scans into detailed segmentations. By training the model on each scan using six hand-segmented image slices out of >1500 in the full leaf scan, it achieves >90% accuracy in background and tissue segmentation. CONCLUSIONS: Overall, this 3D segmentation and quantification pipeline can reduce one of the major barriers to using microCT imaging in high-throughput plant phenotyping.