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Individual plants vary in their ability to respond to environmental changes. The plastic response of a plant enhances its ability to avoid environmental constraints, and hence supports growth, reproduction, and evolutionary and agricultural success.Major progress in the analysis of above- and belowground processes on individual plants has been made by the application of non-invasive imaging methods including Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET).MRI allows for repetitive measurements of roots growing in soil and facilitates quantification of root system architecture traits in 3D. PET, on the other hand, opens a door to analyze dynamic physiological processes in plants such as long-distance carbon transport in a repeatable manner. Combining MRI with PET enables monitoring of short livedCarbon tracer (11C) allocation along the transport paths (i.e. roots visualized by MRI) into active sink structures.To analyse the link between root-internal C allocation patterns and C metabolism in the rhizosphere, we are combining 11CO2 with stable 13CO2 labelling of plants. Isotope ratio mass spectrometry (IRMS) analyses of rhizosphere soil is applied to link root-internal C allocation patterns with distribution of 13C in the rhizosphere soil. The metabolically active rhizosphere organisms are subsequently identified based on DNA 13C stable isotope probing.In our presentation we will highlight our approaches for gathering quantitative data from both image-based technologies in combination with destructive analysis that provides insights into the functioning and dynamics of C transport processes in the plant-soil system.

In natural ecosystems and low-input agriculture systems often the main source of nitrogen is biological nitrogen fixation by symbiotic coexistence with root colonizing microorganisms such as in root nodules in legumes. In return for this nutrient supply, plants allocate significant amount of photosynthetically fixed carbon (C) belowground, fueling activity and growth of the nodules. However, there is still a lack in understanding how plants modulate carbon allocation to a nodulated root system as a dynamic response to abiotic stimuli. Traditional approaches based on destructive sampling make investigations of localized carbon allocation dynamics difficult. Non-destructive 3D-imaging methods including Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) offers new perspective in analysing belowground processes on individual plants. MRI allows for repetitive measurements and quantification of root system architecture traits nodule structures while growing. PET was employed to follow the spatial distribution of leaf-supplied 11 C tracer to nodules and roots. Using Pisum sativum as model for legumes and applying nitrate as an additional N source we investigated short term C allocation dynamics in the root system. We found that the fraction of 11 C tracer arriving in the most active nodules decreased by almost 40% and remained stable between 16h and 42h after the N application. Our results highlight that the combination of MRI-PET enables deeper insights into short term C dynamics of roots and interactions with colonizing microbes. We expect that this modality has high potential for revealing mechanisms that relate to dynamic fitness traits supporting breedingprograms for future crops.

Legumes associate with root colonizing rhizobia that provide fixed nitrogen to its plant host in exchange for recently fixed carbon. There is a lack in understanding how individual plants modulate carbon allocation to a nodulated root system as a dynamic response to abiotic stimuli. One reason is that most approaches are based on destructive sampling, making quantification of localized carbon allocation dynamics in the root system difficult. We established an experimental workflow for routinely using non-invasive Positron Emission Tomography (PET) to follow the allocation of leaf-supplied 11C tracer towards individual nodules in a three-dimensional (3D) root system of pea (Pisum sativum). Nitrate was used for triggering the shutdown of biological nitrogen fixation (BNF) expected to rapidly affect carbon allocation dynamics in the root-nodule system. This nitrate treatment lead to a reduction of 11C tracer allocation to nodules by 40% – 47% in 5 treated plants while the variation in control plants was less than 11%. The established experimental pipeline enabled for the first time that several plants could consistently be labelled and measured using 11C tracer in a PET approach to quantify C-allocation to individual nodules following a BNF shutdown. This demonstrates the strength of using 11C tracers in a PET approach for non-invasive quantification of dynamic carbon allocation in several growing plants over several days. A major advantage of the approach is the possibility to investigate carbon dynamics in small regions of interest in a 3D system such as nodules in comparison to whole plant development.One sentence summaryPositron Emission Tomography for quantification of carbon allocation dynamics in individual nodules within a 3D root system revealed strong effect of nitrate on carbon allocation.

PET imaging is widely used to perform clinical and preclinical studies. A rather new area is the field of plant research. PET allows the dynamic imaging of 3D carbon transport and allocation within plants shoot and root systems of plants. Here, a new system called phenoPET has been developed within the German Plant Phenotyping Network (DPPN) using Digital Photon Counters (DPC) by Philips (digital silicon photo multipliers). Those are organized in horizontal rings forming a Field of View (FOV) of 180mm diameter and 200mm height.The detected hits are arranged in time frames, which are written to disc via USB 3:0 with a maximum transfer rate of 375MBs−1. Preprocessing and coincidence sorting are done offline. To enable high dynamic range phenoPET can drop frames which are not saved on disc when the transfer rate comes to a limit.To compare carbon transport of different plants quantitative images are required. A preliminary normalization and random correction are implemented. In this context, count rate correction and dead time correction are mandatory to avoid quantitative bias. In a decay experiment the dead time of the DPCs reduces the rate of singles by up to 10% at an radioactivity of about 100MBq placed close to the center of the FOV. At this activity about 70% of the frames are dropped leaving a maximum single rate of about 2:5 • 105 cps on each module. Further count rate correction factors for coincidences due to missing frames are presented.

Highlight By employing magnetic resonance imaging to study below-ground sugar beet development, early changes in taproot growth and anatomy could be correlated with the first symptoms of Cercospora leaf spot disease., Cercospora leaf spot (CLS) infection can cause severe yield loss in sugar beet. Introduction of Cercospora-resistant varieties in breeding programmes is important for plant protection to reduce both fungicide applications and the risk of the development of fungal resistance. However, in vivo monitoring of the sugar-containing taproots at early stages of foliar symptoms and the characterization of the temporal development of disease progression has proven difficult. Non-invasive magnetic resonance imaging (MRI) measurements were conducted to quantify taproot development of genotypes with high (HS) and low (LS) levels of susceptibility after foliar Cercospora inoculation. Fourteen days post-inoculation (dpi) the ratio of infected leaf area was still low (~7%) in both the HS and LS genotypes. However, during this period, the volumetric growth of the taproot had already started to decrease. Additionally, inoculated plants showed a reduction of the increase in width of inner cambial rings while the width of outer rings increased slightly compared with non-inoculated plants. This response partly compensated for the reduced development of inner rings that had a vascular connection with Cercospora-inoculated leaves. Hence, alterations in taproot anatomical features such as volume and cambial ring development can be non-invasively detected already at 14 dpi, providing information on the early impact of the infection on whole-plant performance. All these findings show that MRI is a suitable tool to identify promising candidate parent lines with improved resistance to Cercospora, for example with comparatively lower taproot growth reduction at early stages of canopy infection, for future introduction into breeing programmes.

The digitally generated timemarks from more than 2000 individual detector elements in the pheno PET scanner show individual time offsets. These offsets (or skews) are in the range of ± 1 ns while the actual time resolution of each element is below 250 ps. Thus, the time resolution of the entire system will be highly improved by correcting these offsets. This paper presents a precise correction method based on measurements of the LYSObackground radiation. The Coincidence Resolving Time for the entire scanner could be improved from 1.45 ns to less than 300 ps.

Within the German Plant Phenotyping Network (DPPN), we developed a novel PET scanner based on Philips Digital Photon Counters (DPCs, or dSiPMs = digital Silicon Photomultipliers). The scanner is dedicated for plant research and provides functional information on carbon transport within the plant. To this end the detector ring is oriented horizontally. It provides a Field-of-View of 18 cm dia. and 20 cm in height. The read-out electronics cluster hits from different photodetector pixels when they originate from the same scintillation event. These single events are written via USB 3.0 with up to 300 MB/s to the computer system. Crystal identification, energy discrimination and coincidence detection is realized in software. The spatial resolution in the center Field-of-View (CFOV) could be estimated to approx. 1.6 mm from measurements of a dedicated hot rod phantom. Preliminary sensitivity measurements result in a peak sensitivity of 4.04% (ΔE = 250-750 keV) in the CFOV and a Coincidence Resolving Time of 298 ps could be achieved.

Fluxes of mineral nutrients in the xylem are strongly influenced by interactions with the surrounding stem tissues and are probably regulated by them. Toward a mechanistic understanding of these interactions, we applied stable isotope tracers of magnesium, potassium, and calcium continuously to the transpiration stream of cut bean (Phaseolus vulgaris) shoots to study their radial exchange at the cell and tissue level with stem tissues between pith and phloem. For isotope localization, we combined sample preparation with secondary ion mass spectrometry in a completely cryogenic workflow. After 20 min of application, tracers were readily detectable to various degrees in all tissues. The xylem parenchyma near the vessels exchanged freely with the vessels, its nutrient elements reaching a steady state of strong exchange with elements in the vessels within 20 min, mainly via apoplastic pathways. A slow exchange between vessels and cambium and phloem suggested that they are separated from the xylem, parenchyma, and pith, possibly by an apoplastic barrier to diffusion for nutrients (as for carbohydrates). There was little difference in these distributions when tracers were applied directly to intact xylem via a microcapillary, suggesting that xylem tension had little effect on radial exchange of these nutrients and that their movement was mainly diffusive.

A new approach to trace the transport routes of macronutrients in plants at the level of cells and tissues and to measure their elemental distributions was developed for investigating the dynamics and structure-function relationships of transport processes. Stem samples from Phaseolus vulgaris were used as a test system. Shock freezing and cryo-preparation were combined in a cryogenic chain with cryo-time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS) for element and isotope-specific imaging. Cryo-scanning electron microscopy (cryo-SEM) was integrated into the cryogenic workflow to assess the quality of structural preservation. We evaluated the capability of these techniques to monitor transport pathways and processes in xylem and associated tissues using supplementary sodium (Na) and tracers for potassium (K), rubidium (Rb), and 41K added to the transpiration stream. Cryo-ToF-SIMS imaging produced detailed mappings of water, K, calcium, magnesium, the K tracers, and Na without quantification. Lateral resolutions ranged from 10 μm in survey mappings and at high mass resolution to approximately 1 μm in high lateral resolution imaging in reduced areas and at lower mass resolution. The tracers Rb and 41K, as well as Na, were imaged with high sensitivity in xylem vessels and surrounding tissues. The isotope signature of the stable isotope tracer was utilized for relative quantification of the 41K tracer as a fraction of total K at the single pixel level. Cryo-SEM confirmed that tissue structures had been preserved with subcellular detail throughout all procedures. Overlays of cryo-ToF-SIMS images onto the corresponding SEM images allowed detailed correlation of nutrient images with subcellular structures.

Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene–environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure–function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Lateral exchange of water and nutrients between xylem and surrounding tissues helps to de-couple uptake from utilization in all parts of a plant. We studied the dynamics of these exchanges, using stable isotope tracers for water (H(2)(18)O), magnesium ((26)Mg), potassium ((41)K) and calcium ((44)Ca) delivered via a cut stem for various periods to the transpiration stream of bean shoots (Phaseolus vulgaris cv. Fardenlosa Shiny). Tracers were subsequently mapped in stem cross-sections with cryo-secondary ion mass spectrometry. The water tracer equilibrated within minutes across the entire cross-section. In contrast, the nutrient tracers showed a very heterogeneous exchange between xylem vessels and the different stem tissues, even after 4 h. Dynamics of nutrients in the tissues revealed a fast and extensive exchange of nutrients in the xylem parenchyma, with, for example, calcium being completely replaced by tracer in less than 5 min. Dilution of potassium tracer during its 30 s transit in xylem sap through the stem showed that potassium concentration was up-regulated over many hours, to the extent that some of it was probably supplied by phloem recirculation from the shoot.

Lateral exchange of water and nutrients between xylem and surrounding tissues helps to de-couple uptake from utilization in all parts of a plant. We studied the dynamics of these exchanges, using stable isotope tracers for water (H 2 18 O), magnesium ( 26 Mg), potassium ( 41 K) and calcium ( 44 Ca) delivered via a cut stem for various periods to the transpiration stream of bean shoots (Phaseolus vulgaris cv. Fardenlosa Shiny). Tracers were subsequently mapped in stem cross-sections with cryo-secondary ion mass spectrometry. The water tracer equilibrated within minutes across the entire cross-section. In contrast, the nutrient tracers showed a very heterogeneous exchange between xylem vessels and the different stem tissues, even after 4 h. Dynamics of nutrients in the tissues revealed a fast and extensive exchange of nutrients in the xylem parenchyma, with, for example, calcium being completely replaced by tracer in less than 5 min. Dilution of potassium tracer during its 30 s transit in xylem sap through the stem showed that potassium concentration was up-regulated over many hours, to the extent that some of it was probably supplied by phloem recirculation from the shoot.

Background Roots are vital to plants for soil exploration and uptake of water and nutrients. Root performance is critical for growth and yield of plants, in particular when resources are limited. Since roots develop in strong interaction with the soil matrix, tools are required that can visualize and quantify root growth in opaque soil at best in 3D. Two modalities that are suited for such investigations are X-ray Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). Due to the different physical principles they are based on, these modalities have their specific potentials and challenges for root phenotyping. We compared the two methods by imaging the same root systems grown in 3 different pot sizes with inner diameters of 34 mm, 56 mm or 81 mm. Results Both methods successfully visualized roots of two weeks old bean plants in all three pot sizes. Similar root images and almost the same root length were obtained for roots grown in the small pot, while more root details showed up in the CT images compared to MRI. For the medium sized pot, MRI showed more roots and higher root lengths whereas at some spots thin roots were only found by CT and the high water content apparently affected CT more than MRI. For the large pot, MRI detected much more roots including some laterals than CT. Conclusions Both techniques performed equally well for pots with small diameters which are best suited to monitor root development of seedlings. To investigate specific root details or finely graduated root diameters of thin roots, CT was advantageous as it provided the higher spatial resolution. For larger pot diameters, MRI delivered higher fractions of the root systems than CT, most likely because of the strong root-to-soil contrast achievable by MRI. Since complementary information can be gathered with CT and MRI, a combination of the two modalities could open a whole range of additional possibilities like analysis of root system traits in different soil structures or under varying soil moisture. Electronic supplementary material The online version of this article (doi:10.1186/s13007-015-0060-z) contains supplementary material, which is available to authorized users.