Non-destructive determination of floral staging in cereals using X-ray micro computed tomography (µCT)

Background Accurate floral staging is required to aid research into pollen and flower development, in particular male development. Pollen development is highly sensitive to stress and is critical for crop yields. Research into male development under environmental change is important to help target increased yields. This is hindered in monocots as the flower develops internally in the pseudostem. Floral staging studies therefore typically rely on destructive analysis, such as removal from the plant, fixation, staining and sectioning. This time-consuming analysis therefore prevents follow up studies and analysis past the point of the floral staging. Results This study focuses on using X-ray µCT scanning to allow quick and detailed non-destructive internal 3D phenotypic information to allow accurate staging of Arabidopsis thaliana L. and Barley (Hordeum vulgare L.) flowers. X-ray µCT has previously relied on fixation methods for above ground tissue, therefore two contrast agents (Lugol’s iodine and Bismuth) were observed in Arabidopsis and Barley in planta to circumvent this step. 3D models and 2D slices were generated from the X-ray µCT images providing insightful information normally only available through destructive time-consuming processes such as sectioning and microscopy. Barley growth and development was also monitored over three weeks by X-ray µCT to observe flower development in situ. By measuring spike size in the developing tillers accurate non-destructive staging at the flower and anther stages could be performed; this staging was confirmed using traditional destructive microscopic analysis. Conclusion The use of X-ray micro computed tomography (µCT) scanning of living plant tissue offers immense benefits for plant phenotyping, for successive developmental measurements and for accurate developmental timing for scientific measurements. Nevertheless, X-ray µCT remains underused in plant sciences, especially in above-ground organs, despite its unique potential in delivering detailed non-destructive internal 3D phenotypic information. This work represents a novel application of X-ray µCT that could enhance research undertaken in monocot species to enable effective non-destructive staging and developmental analysis for molecular genetic studies and to determine effects of stresses at particular growth stages

Investigating the microstructure of plant leaves in 3D with lab-based X-ray Computed Tomography

Background Leaf cellular architecture plays an important role in setting limits for carbon assimilation and, thus, photosynthetic performance. However, the low density, fine structure, and sensitivity to desiccation of plant tissue has presented challenges to its quantification. Classical methods of tissue fixation and embedding prior to 2D microscopy of sections is both laborious and susceptible to artefacts that can skew the values obtained. Here we report an image analysis pipeline that provides quantitative descriptors of plant leaf intercellular airspace using lab-based X-ray Computed Tomography (microCT). We demonstrate successful visualisation and quantification of differences in leaf intercellular airspace in 3D for a range of species (including both dicots and monocots) and provide a comparison with a standard 2D analysis of leaf sections. Results We used the microCT image pipeline to obtain estimates of leaf porosity and mesophyll exposed surface area (Smes) for three dicot species (Arabidopsis, tomato and pea) and three monocot grasses (barley, oat and rice). The imaging pipeline consisted of (1) a masking operation to remove the background airspace surrounding the leaf, (2) segmentation by an automated threshold in ImageJ and then (3) quantification of the extracted pores using the ImageJ ‘Analyze Particles’ tool. Arabidopsis had the highest porosity and lowest Smes for the dicot species whereas barley had the highest porosity and the highest Smes for the grass species. Comparison of porosity and Smes estimates from 3D microCT analysis and 2D analysis of sections indicates that both methods provide a comparable estimate of porosity but the 2D method may underestimate Smes by almost 50%. A deeper study of porosity revealed similarities and differences in the asymmetric distribution of airspace between the species analysed. Conclusions Our results demonstrate the utility of high resolution imaging of leaf intercellular airspace networks by lab-based microCT and provide quantitative data on descriptors of leaf cellular architecture. They indicate there is a range of porosity and Smes values in different species and that there is not a simple relationship between these parameters, suggesting the importance of cell size, shape and packing in the determination of cellular parameters proposed to influence leaf photosynthetic performance

Partner choice through concealed floral sugar rewards evolved with the specialization of ant-plant mutualisms.

SummaryObligate mutualisms require filtering mechanisms to prevent their exploitation by opportunists, but ecological contexts and traits facilitating the evolution of such mechanisms are largely unknown. We investigated the evolution of filtering mechanisms in an epiphytic ant-plant symbiotic system in Fiji involving Rubiaceae and dolichoderine ants, using field experiments, metabolomics, X-ray micro-computed tomography (micro-CT) scanning and phylogenetics. We discovered a novel plant reward consisting of sugary sap concealed in post-anthetic flowers only accessible to Philidris nagasau workers that bite through the thick epidermis. In five of the six species of Rubiaceae obligately inhabited by this ant, the nectar glands functioned for 10 d after a flowerandapos;s sexual function was over. Sugar metabolomics and field experiments showed that ant foraging tracks sucrose levels, which only drop at the onset of fruit development. Ontogenetic analyses of our focal species and their relatives revealed a 25-fold increase in nectary size and delayed fruit development in the ant-rewarding species, and Bayesian analyses of several traits showed the correlated evolution of sugar rewards and symbiosis specialization. Concealed floral nectar forestalls exploitation by opportunists (generalist ants) and stabilizes these obligate mutualisms. Our study pinpoints the importance of partner choice mechanisms in transitions from facultative to obligate mutualisms.</p

Partner choice through concealed floral sugar rewards evolved with the specialization of ant-plant mutualisms.

SummaryObligate mutualisms require filtering mechanisms to prevent their exploitation by opportunists, but ecological contexts and traits facilitating the evolution of such mechanisms are largely unknown. We investigated the evolution of filtering mechanisms in an epiphytic ant-plant symbiotic system in Fiji involving Rubiaceae and dolichoderine ants, using field experiments, metabolomics, X-ray micro-computed tomography (micro-CT) scanning and phylogenetics. We discovered a novel plant reward consisting of sugary sap concealed in post-anthetic flowers only accessible to Philidris nagasau workers that bite through the thick epidermis. In five of the six species of Rubiaceae obligately inhabited by this ant, the nectar glands functioned for 10 d after a flower&amp;apos;s sexual function was over. Sugar metabolomics and field experiments showed that ant foraging tracks sucrose levels, which only drop at the onset of fruit development. Ontogenetic analyses of our focal species and their relatives revealed a 25-fold increase in nectary size and delayed fruit development in the ant-rewarding species, and Bayesian analyses of several traits showed the correlated evolution of sugar rewards and symbiosis specialization. Concealed floral nectar forestalls exploitation by opportunists (generalist ants) and stabilizes these obligate mutualisms. Our study pinpoints the importance of partner choice mechanisms in transitions from facultative to obligate mutualisms.</p