Ralf Metzner

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.

Imaging Nutrient Distributions in Plant Tissue Using Time-of-Flight Secondary Ion Mass Spectrometry and Scanning Electron Microscopy

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.

Contrasting dynamics of water and mineral nutrients in stems shown by stable isotope tracers and cryo‐SIMS

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.

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.

Tracing cationic nutrients from xylem into stem tissue of French bean by stable isotope tracers and cryo-secondary ion mass spectrometry.

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.

Belowground plant development measured with magnetic resonance imaging (MRI): exploiting the potential for non-invasive trait quantification using sugar beet as a proxy

Both structural and functional properties of belowground plant organs are critical for the development and yield of plants but, compared to the shoot, much more difficult to observe due to soil opacity. Many processes concerning the belowground plant performance are not fully understood, in particular spatial and temporal dynamics and their interrelation with environmental factors. We used Magnetic Resonance Imaging (MRI) as a noninvasive method to evaluate which traits can be measured when a complex plant organ is monitored in-vivo while growing in the soil. We chose sugar beet (Beta vulgaris ssp. vulgaris) as a model system. The beet consists mainly of root tissues, is rather complex regarding tissue structure and responses to environmental factors, and thereby a good object to test the applicability of MRI for 3D phenotyping approaches. Over a time period of up to 3 months, traits such as beet morphology or anatomy were followed in the soil and the effect of differently sized pots on beet fresh weight calculated from MRI data was studied. There was a clear positive correlation between the pot size and the increase in fresh weight of a sugar beet over time. Since knowledge of the development of internal beet structures with several concentric cambia, vascular and parenchyma rings is still limited, we consecutively acquired 3D volumetric images on individual plants using the MRI contrast parameter T2 to map the development of rings at the tissue level. This demonstrates that MRI provides versatile protocols to non-invasively measure plant traits in the soil. It opens new avenues to investigate belowground plant performance under adverse environmental conditions such as drought, nutrient shortage, or soil compaction to seek for traits of belowground organs making plants more resilient to stress.

Magnetic resonance imaging of sugar beet taproots in soil reveals growth reduction and morphological changes during foliar Cercospora beticola infestation

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.