Read-out electronics for digital silicon photomultiplier modules

A new kind of a PET-Scanner (PET = positron emission tomography) for plant research is developed asa joint project of the Forschungszentrum Jülich and Philips Digital Photon Counting (PDPC). Thisscanner will utilize digital silicon photomultiplier (dSiPM) for plant phenotyping for the very first time.The goal of this work is to get a further knowledge of the operation of digital silicon photomultiplier.On this account a test-facility for this new photo detectors has been installed at the central instituteof engineering, electronics and analytics (ZEA-2 electronic systems) to determine the usage of thissensors, having regard to use them as scintillation detectors in a PET-Scanner later on.This work has its focus on the development of a fast read-out electronic for the used photo sensorsDPC3200-22-44. As there will be high data rates a fast USB 3.0 interface has been used. All thenecessary processing and data handling has been implemented in a state of the art FPGA

Development Of A Gas Exchange System To Adminster Radioactive Carbon Dioxide 11CO2 To Plants

Positron Emission Tomography (PET) is a non-invasive method and a key technology to study functional root traits of different plant species or genotypes. It allows monitoring transport processes in plants consecutively over a longer period of time as well as plant reactions to environmental changes or induced stresses in vivo. Radioactive carbon dioxide, which is labelled with the positron emitting 11C (half-life: 20 min), can be administered to a plant to follow up and quantify CO2 uptake as well as transport and storage of the labeled photoassimilates in particular in the roots without harming the plants. Since in the experiment radioactive tracer is handled, specific safety precautions have to be met. We have developed a prototype of a gas exchange system that enables measuring of gas exchange rates (CO2 and water) of a plant and, at the same time, ensures safe administration and discharge of the radioactive 11CO2 during the measurements with the PET system. Initially, the gas exchange system provides a certain gas composition in which both CO2 concentration and humidity can be adjusted. Under the control of mass flow rate (range 0.5 to 3.0 L/min) and differential pressure towards atmosphere the gas is delivered to the plant, which is enclosed in a glass cuvette. An infrared gas analyzer detects the delta CO2 and H2O concentrations between the gas flow entering and leaving the plant cuvette with an accuracy of +/-1 µmol/mol or mmol/mol, respectively. For the administration of the radioactive species of CO2 to the plant, the system can be operated in a closed cycle by which the plant will be exposed to a certain amount of 11CO2 for short time (seconds to minutes). After the pulse labeling is completed, the CO2 together with the radioactive 11CO2 will be adsorbed on the outlet of the system for safe discharge. We are currently evaluating the prototype system under real test conditions for adaptation and optimization. Future work will include automation of the 11CO2 administration and gas exchange measurements

Labelling plants with radioactive 11CO2 for noninvasive 3D imaging with PET

Plant carbon dioxide fixation and subsequent photoassimilates allocation are fundamentally important for survival, growth and yield of plants. Since carbon distribution in a plant is highly dynamic its investigation is a unique challenge. Radiotracers, such as radioactive carbon dioxide 11CO2 can be administered to a leaf or canopy for tracing photoassimilate distribution within a plant. The 3D distribution of the 11C tracer can be monitored with a positron emission tomograph (PET) in order to obtain carbon transport parameters for functional phenotyping. For labelling plants we established a gas exchange system for both measuring gas exchange of leaves and administering 11CO2 to the plant. Handling the radioactive carbon dioxide safely requires special precautions which are implemented in the system. Here, we present results of the first gas exchange measurements of pea (Pisum sativum) under drought stress as well as images of 11C allocation into the root measured with the PET system ‘PlanTIS’. For an improved 3D visualization of 11C transport a new PET system (phenoPET) was constructed, which is currently under evaluation. First experimental results on plants with both phenoPET and the gas exchange system are expected by mid of 2016. In future, it is planned to automatize plant transport, labeling and tracer measurement. All installations combined will facilitate dynamic monitoring and quantification of carbon assimilation with regards to different phenotypes and under controlled environmental conditions

Whole plant 13CO2-labelling for carotenoid turnover analysis in leaves

Understanding the regulation of carotenoid metabolism (synthesis, conversion and degradation) in plants requires turnover measurement of individual carotenoids, apocarotenoids and precursors. In our previous study, we demonstrated continuous turnover of carotenes together with chlorophyll a in illuminated leaves of Arabidopsis thaliana by using 14CO2 pulse-chase labeling. In contrast to carotenes and chlorophyll a that are bound in photosystem reaction center complexes, xanthophylls and chlorophyll b, which are bound in light-harvesting antenna complexes, were hardly labeled by 14C within a day, even when the total amount of xanthophyll-cycle pigments (zeaxanthin, antheraxanthin, and violaxanthin) was increasing, presumably by de novo synthesis, under strong light. In order to obtain quantitative information of carotenoid turnover in leaves of intact plants, we constructed a labelling chamber in which 15 small plants, such as Arabidopsis, can be synchronously labelled by 13CO2 over days. First we tested the chamber by operating with 12CO2 while continuously monitoring the conditions inside the chamber (light intensity, air temperature and humidity, CO2 concentration, pressure). Then a protocol was established to grow 15 Arabidopsis plants (wildtype Columbia-0) in the chamber under the light intensity of ~200 µmol photons m-2 s-1. Switching to 13CO2, plants were grown in the chamber under the same conditions for up to seven days. LC-MS analysis of pigments extracted from rosette leaves of the 13C-labelled Arabidopsis plants showed a substantial incorporation of photosynthetically fixed 13C into β-carotene and lutein along with chlorophyll a and chlorophyll b. The proposed system can be used for pulse-chase experiments (from 12CO2 to 13CO2 and vice versa) to estimate the turnover rate of carotenoids and chlorophylls as well as other plant metabolites. Results from such experiments could provide missing pieces of information in the current picture of metabolic pathway regulation in plants

In vivo monitoring of legume root and nodule development

Quantitative non-invasive measurement of structural and functional development of plant organs allows for deep phenotyping and dynamic investigation of plant performance under stress. While this can be done straightforward for leaves or stems other plant parts, such as seeds enclosed in pods or roots and nodules hidden in soil are more difficult to investigate. Their development however is critical for yield and performance under stress and direct observations in conjunction with genetic and metabolomics approaches may hint on the underlying mechanisms. Here, we apply a set of three non-invasive techniques for studying such developmental processes: 1) Low field nuclear magnetic resonance relaxometry with portable devices (pNMR) is used to study dry matter and water content in pods over periods of several weeks. 2) Magnetic Resonance Imaging (MRI) is used to study the structural development of roots and nodules in soil filled pots over several weeks. 3) Positron Emission Tomography (PET) with the short-lived radiotracer 11C is used to monitor the partitioning of photoassimilates and its dynamics among roots and nodules. We show the application of all three techniques to pea and bean plants grown in soil. We also discuss their potential to provide a direct view on the effects of genotype or rhizobial strain on plant performance under stress and on biological nitrogen fixation

Noninvasive 3D Root Imaging

The influence of roots on plant productivity has often been neglected because of the difficulties to access and monitor the root system architecture and function. The goals of this work are to establish methods to noninvasively image 3D root system architecture (RSA) in 3D, to identify structural and functional root traits, to monitor the development of plant root traits during development and, in particular, to identify traits of resource efficient roots. Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) are two modalities which enable observing structural and functional properties of roots growing in soil in a noninvasive manner. The existing 4.7T MRI System has been shown to produce 3D images with a high root to soil contrast [1]. Due to the installed prototypic robot system these data sets can be acquired automatically, including measurements during the night and on weekends, leading to a considerable amount of raw data. To enable calculation of RSA traits and their development over time, a software tool has been developed capable of extracting the RSA from the MRI measurement data automatically. Methods to manually correct the automatically extracted RSA have been implemented. Typical root traits calculated from the extracted RSA are shown, including a comparison to an invasive method (WinRhizo).Functional information, in particular of carbon transport, of intact root systems can be obtained by positron emission tomography (PET). Radioactively labelled [11C]-CO2 is taken up by photosynthesis and radiolabelled metabolites (tracer) are eventually transported into the root system. The existing PET system (PlanTIS [1]) is used for test experiments though its detection sensitivity is too low to characterize transport properties. To overcome the drawbacks of PlanTIS, a new PET system (phenoPET) has been developed together with Philips Photon Counting and two institutes at Forschungszentrum Jülich (ZEA-1 and ZEA-2). The phenoPET is currently being assembled and will be delivered in 2015. Compared to PlanTIS, the new phenoPET system will provide higher sensitivity and a larger field of view, two important factors to enable functional phenotyping.Literature:[1] Jahnke et al.: Combined MRI–PET dissects dynamic changes in plant structures and functions. The Plant Journal (2009) 59, 634–64

Combination of long-term 13CO2 labeling and isotopolog profiling allows turnover analysis of photosynthetic pigments in Arabidopsis leaves

BACKGROUND: Living cells maintain and adjust structural and functional integrity by continual synthesis and degradation of metabolites and macromolecules. The maintenance and adjustment of thylakoid membrane involve turnover of photosynthetic pigments along with subunits of protein complexes. Quantifying their turnover is essential to understand the mechanisms of homeostasis and long-term acclimation of photosynthetic apparatus. Here we report methods combining whole-plant long-term (13)CO(2) labeling and liquid chromatography - mass spectrometry (LC–MS) analysis to determine the size of non-labeled population (NLP) of carotenoids and chlorophylls (Chl) in leaf pigment extracts of partially (13)C-labeled plants. RESULTS: The labeling chamber enabled parallel (13)CO(2) labeling of up to 15 plants of Arabidopsis thaliana with real-time environmental monitoring ([CO(2)], light intensity, temperature, relative air humidity and pressure) and recording. No significant difference in growth or photosynthetic pigment composition was found in leaves after 7-d exposure to normal CO(2) (~ 400 ppm) or (13)CO(2) in the labeling chamber, or in ambient air outside the labeling chamber (control). Following chromatographic separation of the pigments and mass peak assignment by high-resolution Fourier-transform ion cyclotron resonance MS, mass spectra of photosynthetic pigments were analyzed by triple quadrupole MS to calculate NLP. The size of NLP remaining after the 7-d (13)CO(2) labeling was ~ 10.3% and ~ 11.5% for all-trans- and 9-cis-β-carotene, ~ 21.9% for lutein, ~ 18.8% for Chl a and 33.6% for Chl b, highlighting non-uniform turnover of these pigments in thylakoids. Comparable results were obtained in all replicate plants of the (13)CO(2) labeling experiment except for three that were showing anthocyanin accumulation and growth impairment due to insufficient water supply (leading to stomatal closure and less (13)C incorporation). CONCLUSIONS: Our methods allow (13)CO(2) labeling and estimation of NLP for photosynthetic pigments with high reproducibility despite potential variations in [(13)CO(2)] between the experiments. The results indicate distinct turnover rates of carotenoids and Chls in thylakoid membrane, which can be investigated in the future by time course experiments. Since (13)C enrichment can be measured in a range of compounds, long-term (13)CO(2) labeling chamber, in combination with appropriate MS methods, facilitates turnover analysis of various metabolites and macromolecules in plants on a time scale of hours to days. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-022-00946-3

Harnessing the power of 11C-labelling and Positron Emission Tomography (PET) for investigating Phloem velocities above and belowground

The short-lived radioisotope 11C can be applied non-invasively to the plant as 11CO2 to follow the flow of recently fixed carbon. This method has allowed for many interesting findings on phloem flow in the past. The combination with PET detection and compartmental modelling has the potential to allow the imaging and quantification of phloem flow in complex 3D structures such as root system or branched shoots. However, this requires an experimental pipeline and facility to label and image plants in a reliable and consistent manner. We will show the key elements of the pipeline we have established in a plant-dedicated radiotracer lab for routine flow imaging along with discussing the advantages and limitation of the approach. Results will be presented on phloem flow velocities simultaneously measured in different root types of maize with statistically relevant numbers of individuals and other 3D examples. Furthermore, results will be presented on phloem flow in different parts of bean shoots and examples for other species