Precision Phenotyping of Nectar-Related Traits Using X-ray Micro Computed Tomography.

Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant-pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.

Advances on the Visualization of the Internal Structures of the European Mistletoe: 3D Reconstruction Using Microtomography.

The European mistletoe (Viscum album) is a dioecious epiphytic evergreen hemiparasite that develops an extensive endophyte enabling the absorption of water and mineral salts from the host tree, whereas the exophytic leaves are photosynthetically active. The attachment mode and host penetration are well studied, but little information is available about the effects of mistletoe age and sex on haustorium-host interactions. We harvested 130 plants of Viscum album ssp. album growing on host branches of Aesculus flava for morphological and anatomical investigations. Morphometric analyses of the mistletoe and the (hypertrophied) host interaction site were correlated with mistletoe age and sex. We recorded the morphology of the endophytic systems of various ages by using X-ray microtomography scans and corresponding stereomicroscopic images. For detailed anatomical studies, we examined thin stained sections of the mistletoe-host interface by light microscopy. The diameter and length of the branch hypertrophy showed a positive linear correlation with the age of the mistletoe. Correlations with their sex were only found for ratios between host branch and hypertrophy size. A female bias of about 76% was found. In a 4-year-old mistletoe, several small, almost equally sized sinkers and the connected cortical strands extend over more than 5 cm within the host branch. In older mistletoes, one main sinker was predominant and occupied an increasingly large proportion of the stem cross-section. Bands of vessels ran along the axis of the wedge-shaped haustoria and sinkers and bent sideways toward the mistletoe-host interface. At the interface, the vascular elements of the host wood changed their direction and formed vortices near the haustorium.

Visualization of xylem embolism by X-ray microtomography: a direct test against hydraulic measurements.

X-ray microtomography (microCT) is becoming a valuable noninvasive tool for advancing our understanding of plant-water relations. Laboratory-based microCT systems are becoming more affordable and provide better access than synchrotron facilities. However, some systems come at the cost of comparably lower signal quality and spatial resolution than synchrotron facilities. In this study, we evaluated laboratory-based X-ray microCT imaging as a tool to nondestructively analyse hydraulic vulnerability to drought-induced embolism in a woody plant species. We analysed the vulnerability to drought-induced embolism of benchtop-dehydrated Eucalyptus camaldulensis plants using microCT and hydraulic flow measurements on the same sample material, allowing us to directly compare the two methods. Additionally, we developed a quantitative procedure to improve microCT image analysis at limited resolution and accurately measure vessel lumens. Hydraulic measurements matched closely with microCT imaging of the current-year growth ring, with similar hydraulic conductivity and loss of conductivity due to xylem embolism. Optimized thresholding of vessel lumens during image analysis, based on a physiologically meaningful parameter (theoretical conductivity), allowed us to overcome common potential constraints of some lab-based systems. Our results indicate that estimates of vulnerability to embolism provided by microCT visualization agree well with those obtained from hydraulic measurements on the same sample material.

X-ray microtomography observations of xylem embolism in stems of Laurus nobilis are consistent with hydraulic measurements of percentage loss of conductance.

Drought-induced xylem embolism is a serious threat to plant survival under future climate scenarios. Hence, accurate quantification of species-specific vulnerability to xylem embolism is a key to predict the impact of climate change on vegetation. Low-cost hydraulic measurements of embolism rate have been suggested to be prone to artefacts, thus requiring validation by direct visualization of the functional status of xylem conduits using nondestructive imaging techniques, such as X-ray microtomography (microCT). We measured the percentage loss of conductance (PLC) of excised stems of Laurus nobilis (laurel) dehydrated to different xylem pressures, and compared results with direct observation of gas-filled vs water-filled conduits at a synchrotron-based microCT facility using a phase contrast imaging modality. Theoretical PLC calculated on the basis of microCT observations in stems of laurel dehydrated to different xylem pressures overall were in agreement with hydraulic measurements, revealing that this species suffers a 50% loss of xylem hydraulic conductance at xylem pressures averaging -3.5 MPa. Our data support the validity of estimates of xylem vulnerability to embolism based on classical hydraulic techniques. We discuss possible causes of discrepancies between data gathered in this study and those of recent independent reports on laurel hydraulics.