Siegfried Jahnke

Combined MRI–PET dissects dynamic changes in plant structures and functions

Unravelling the factors determining the allocation of carbon to various plant organs is one of the great challenges of modern plant biology. Studying allocation under close to natural conditions requires non-invasive methods, which are now becoming available for measuring plants on a par with those developed for humans. By combining magnetic resonance imaging (MRI) and positron emission tomography (PET), we investigated three contrasting root/shoot systems growing in sand or soil, with respect to their structures, transport routes and the translocation dynamics of recently fixed photoassimilates labelled with the short-lived radioactive carbon isotope (11)C. Storage organs of sugar beet (Beta vulgaris) and radish plants (Raphanus sativus) were assessed using MRI, providing images of the internal structures of the organs with high spatial resolution, and while species-specific transport sectoralities, properties of assimilate allocation and unloading characteristics were measured using PET. Growth and carbon allocation within complex root systems were monitored in maize plants (Zea mays), and the results may be used to identify factors affecting root growth in natural substrates or in competition with roots of other plants. MRI-PET co-registration opens the door for non-invasive analysis of plant structures and transport processes that may change in response to genomic, developmental or environmental challenges. It is our aim to make the methods applicable for quantitative analyses of plant traits in phenotyping as well as in understanding the dynamics of key processes that are essential to plant performance.

Temperature responses of roots: impact on growth, root system architecture and implications for phenotyping

Root phenotyping is a challenging task, mainly because of the hidden nature of this organ. Only recently, imaging technologies have become available that allow us to elucidate the dynamic establishment of root structure and function in the soil. In root tips, optical analysis of the relative elemental growth rates in root expansion zones of hydroponically-grown plants revealed that it is the maximum intensity of cellular growth processes rather than the length of the root growth zone that control the acclimation to dynamic changes in temperature. Acclimation of entire root systems was studied at high throughput in agar-filled Petri dishes. In the present study, optical analysis of root system architecture showed that low temperature induced smaller branching angles between primary and lateral roots, which caused a reduction in the volume that roots access at lower temperature. Simulation of temperature gradients similar to natural soil conditions led to differential responses in basal and apical parts of the root system, and significantly affected the entire root system. These results were supported by first data on the response of root structure and carbon transport to different root zone temperatures. These data were acquired by combined magnetic resonance imaging (MRI) and positron emission tomography (PET). They indicate acclimation of root structure and geometry to temperature and preferential accumulation of carbon near the root tip at low root zone temperatures. Overall, this study demonstrated the value of combining different phenotyping technologies that analyse processes at different spatial and temporal scales. Only such an integrated approach allows us to connect differences between genotypes obtained in artificial high throughput conditions with specific characteristics relevant for field performance. Thus, novel routes may be opened up for improved plant breeding as well as for mechanistic understanding of root structure and function.

Imaging plants dynamics in heterogenic environments.

Noninvasive imaging sensors and computer vision approaches are key technologies to quantify plant structure, physiological status, and performance. Today, imaging sensors exploit a wide range of the electromagnetic spectrum, and they can be deployed to measure a growing number of traits, also in heterogenic environments. Recent advances include the possibility to acquire high-resolution spectra by imaging spectroscopy and classify signatures that might be informative of plant development, nutrition, health, and disease. Three-dimensional (3D) reconstruction of surfaces and volume is of particular interest, enabling functional and mechanistic analyses. While taking pictures is relatively easy, quantitative interpretation often remains challenging and requires integrating knowledge of sensor physics, image analysis, and complex traits characterizing plant phenotypes.

Non-invasive approaches for phenotyping of enhanced performance traits in bean

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.

Continuous Turnover of Carotenes and Chlorophyll a in Mature Leaves of Arabidopsis Revealed by 14CO2 Pulse-Chase Labeling

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by 14CO2 pulse-chase labeling under control-light (CL; 130 μmol photons m−2 s−1) and high-light (HL; 1,000 μmol photons m−2 s−1) conditions. Following a 30-min 14CO2 administration, photosynthetically fixed 14C was quickly incorporated in β-carotene (β-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, 14C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the 14C fractions incorporated in β-C and Chl a, whereas long-term HL acclimation significantly enhanced 14C labeling of Chl a but not β-C. Consequently, the maximal 14C signal ratio between β-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing α-carotene (α-C) together with reduced amounts of β-C, remarkably high 14C labeling was found for α-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum 14C ratios between carotenes and Chl a were 1:2 for α-C:Chl a and 1:5 for β-C:Chl a in CL-grown lut5 plants, suggesting high turnover of α-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.

MRI links stem water content to stem diameter variations in transpiring trees

In trees, stem diameter variations are related to changes in stem water content, because internally stored water is depleted and replenished over a day. To confirm this relationship, non-invasive magnetic resonance imaging (MRI) was combined with point dendrometer measurements in three actively transpiring oak (Quercus robur L.) trees. Two of these oak trees were girdled to study the stem increment above the girdling zone. MRI images and micrographs of stem cross-sections revealed a close link between the water distribution and the anatomical features of the stem. Stem tissues with the highest amount of water were physiologically the most active ones, being the youngest differentiating xylem cells, the cambium and the youngest differentiating and conductive phloem cells. Daily changes in stem diameter corresponded well with the simultaneously MRI-measured amount of water, confirming their strong interdependence. MRI images also revealed that the amount of water in the elastic bark tissues, excluding cambium and the youngest phloem, contributed most to the daily stem diameter changes. After bark removal, an additional increase in stem diameter was measured above the girdle. This increase was attributed not only to the cambial production of new cells, but also to swelling of existing bark cells. In conclusion, the comparison of MRI and dendrometer measurements confirmed previous interpretations and applications of dendrometers and illustrates the additional and complementary information MRI can reveal regarding water relations in plants.

Design and initial performance of PlanTIS: a high-resolution positron emission tomograph for plants

Positron emitters such as (11)C, (13)N and (18)F and their labelled compounds are widely used in clinical diagnosis and animal studies, but can also be used to study metabolic and physiological functions in plants dynamically and in vivo. A very particular tracer molecule is (11)CO(2) since it can be applied to a leaf as a gas. We have developed a Plant Tomographic Imaging System (PlanTIS), a high-resolution PET scanner for plant studies. Detectors, front-end electronics and data acquisition architecture of the scanner are based on the ClearPET system. The detectors consist of LSO and LuYAP crystals in phoswich configuration which are coupled to position-sensitive photomultiplier tubes. Signals are continuously sampled by free running ADCs, and data are stored in a list mode format. The detectors are arranged in a horizontal plane to allow the plants to be measured in the natural upright position. Two groups of four detector modules stand face-to-face and rotate around the field-of-view. This special system geometry requires dedicated image reconstruction and normalization procedures. We present the initial performance of the detector system and first phantom and plant measurements.

11C-PET imaging reveals transport dynamics and sectorial plasticity of oak phloem after girdling

Carbon transport processes in plants can be followed non-invasively by repeated application of the short-lived positron-emitting radioisotope 11C, a technique which has rarely been used with trees. Recently, positron emission tomography (PET) allowing 3D visualization has been adapted for use with plants. To investigate the effects of stem girdling on the flow of assimilates, leaves on first order branches of two-year-old oak (Quercus robur L.) trees were labeled with 11C by supplying 11CO2-gas to a leaf cuvette. Magnetic resonance imaging gave an indication of the plant structure, while PET registered the tracer flow in a stem region downstream from the labeled branches. After repeated pulse labeling, phloem translocation was shown to be sectorial in the stem: leaf orthostichy determined the position of the phloem sieve tubes containing labeled 11C. The observed pathway remained unchanged for days. Tracer time-series derived from each pulse and analysed with a mechanistic model showed for two adjacent heights in the stem a similar velocity but different loss of recent assimilates. With either complete or partial girdling of bark within the monitored region, transport immediately stopped and then resumed in a new location in the stem cross-section, demonstrating the plasticity of sectoriality. One day after partial girdling, the loss of tracer along the interrupted transport pathway increased, while the velocity was enhanced in a non-girdled sector for several days. These findings suggest that lateral sugar transport was enhanced after wounding by a change in the lateral sugar transport path and the axial transport resumed with the development of new conductive tissue.

Root-root interactions: extending our perspective to be more inclusive of the range of theories in ecology and agriculture using in-vivo analyses

BACKGROUND There is a large body of literature on competitive interactions among plants, but many studies have only focused on above-ground interactions and little is known about root-root dynamics between interacting plants. The perspective on possible mechanisms that explain the outcome of root-root interactions has recently been extended to include non-resource-driven mechanisms (as well as resource-driven mechanisms) of root competition and positive interactions such as facilitation. These approaches have often suffered from being static, partly due to the lack of appropriate methodologies for in-situ non-destructive root characterization. SCOPE Recent studies show that interactive effects of plant neighbourhood interactions follow non-linear and non-additive paths that are hard to explain. Common outcomes such as accumulation of roots mainly in the topsoil cannot be explained solely by competition theory but require a more inclusive theoretical, as well as an improved methodological framework. This will include the question of whether we can apply the same conceptual framework to crop versus natural species. CONCLUSIONS The development of non-invasive methods to dynamically study root-root interactions in vivo will provide the necessary tools to study a more inclusive conceptual framework for root-root interactions. By following the dynamics of root-root interactions through time in a whole range of scenarios and systems, using a wide variety of non-invasive methods, (such as fluorescent protein which now allows us to separately identify the roots of several individuals within soil), we will be much better equipped to answer some of the key questions in root physiology, ecology and agronomy.

Quantitative 3D Analysis of Plant Roots Growing in Soil Using Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) enables nondestructive 3D imaging and quantification of roots or root system architecture in soil and is suited for automated and routine measurements of root development. Precise measurements of root system architecture traits are an important requirement for plant phenotyping. Most of the current methods for analyzing root growth require either artificial growing conditions (e.g. hydroponics), are severely restricted in the fraction of roots detectable (e.g. rhizotrons), or are destructive (e.g. soil coring). On the other hand, modalities such as magnetic resonance imaging (MRI) are noninvasive and allow high-quality three-dimensional imaging of roots in soil. Here, we present a plant root imaging and analysis pipeline using MRI together with an advanced image visualization and analysis software toolbox named NMRooting. Pots up to 117 mm in diameter and 800 mm in height can be measured with the 4.7 T MRI instrument used here. For 1.5 l pots (81 mm diameter, 300 mm high), a fully automated system was developed enabling measurement of up to 18 pots per day. The most important root traits that can be nondestructively monitored over time are root mass, length, diameter, tip number, and growth angles (in two-dimensional polar coordinates) and spatial distribution. Various validation measurements for these traits were performed, showing that roots down to a diameter range between 200 μm and 300 μm can be quantitatively measured. Root fresh weight correlates linearly with root mass determined by MRI. We demonstrate the capabilities of MRI and the dedicated imaging pipeline in experimental series performed on soil-grown maize (Zea mays) and barley (Hordeum vulgare) plants.