Arnd J. Kuhn

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.

Monitoring uptake and contents of Mg, Ca and K in Norway spruce as influenced by pH and Al, using microprobe analysis and stable isotope labelling

In a model system using intact spruce trees (Picea abies [L.] Karst.) we followed the path of magnesium, calcium and potassium during uptake into the root and during long-range transport into the shoot, by multiple stable isotope labelling. The roots of two- and three-year-old spruce trees originating from soil culture were removed from the soil and, in part or in toto, exposed to labelling solutions containing the stable isotopes 25Mg or 26Mg, 41K and 42Ca or 44Ca. Optical-emission-spectroscopy (ICP-OES) of plant fractions and labelling solutions was combined with the quantitative analysis of stable isotope ratios in sections of shock frozen, cryosubstituted material using the laser-microprobe-mass-analyser (LAMMA). This combination allowed us to distinguish, both in bulk samples and on the cellular level between (i) the fraction of elements originally present in the plant before the start of the labelling, (ii) the material taken up from the labelling solution into the plant and (iii) any material released by the plant into the labelling solution.In single-root labelling experiments, roots of three-year-old spruce trees, grown in nursery soil, were exposed to various pH conditions. The exchange of Mg and Ca with the labelling solution was nearly 100% in the cell walls of the mycorrhized finest roots. This exchange was only slightly affected by a step down to pH 3.5. The absolute Mg and Ca content in the cell walls was moderately reduced by incubation at pH 3.5 and strongly reduced in the presence of Al at this pH. After a pH 3.5 and 2 mM Al treatment we found Al in the xylem cell walls and the cortex cell lumina at elevated concentrations. To analyse the combined effect of high Al and high proton concentrations on the long-range transport, we used a “split-root system”. The root mass of an intact two-year-old spruce tree, grown in mineral soil, was divided into even parts and both halves incubated in solutions with two sets of different stable isotopes of Mg and Ca (side A: no Al, 25Mg and 42Ca; side B: +Al, 26Mg and 44Ca) and 41K on both sides. We observed a large uptake of Mg, Ca and K into the plant and a pronounced release. The net uptake of all three elements was lower from the Al-doted solution. In cross-sections of the apical shoot we found after seven-day labelling period about 60–70% of the Mg and Ca and 30% of the K content in the xylem cell walls originating from both labelling solutions. The clear majority of the Mg and Ca label originated from the Al-doted side.

The kinetics of calcium and magnesium entry into mycorrhizal spruce roots

Abstract. The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Piceaabies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers 25Mg and 44Ca. After labelling, samples of roots were excised from the plants, shock-frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t1/2 of 100–120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While 25Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at +6 °C were similar to those obtained at a root temperature of +22 °C.

The mycorrhizal fungus Paxillus involutus transports magnesium to Norway spruce seedlings. Evidence from stable isotope labeling

Although it is well established that ectomycorrhizas improve the mineral nutrition of forest trees, there has been little evidence that they mediate uptake of divalent cations such as Mg. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus Batsch in a sand culture system with two compartments separated by a 45-μm Nylon mesh. Hyphae, but not roots, can penetrate this net. Labeling the compartment only accessible to hyphae with 25Mg showed that hyphae of the ectomycorrhizal fungus Paxillus involutus transported Mg to their host plant. No label was found in nonmycorrhizal control plants. Our data support the idea that ectomycorrhizas are important for the Mg nutrition of forest trees.

Localisation of Aluminium in Root Tips of Zea mays and Vicia faba

Summary Monocot species and dicot species differ widely in the composition of their cell walls, namely in the pectin content, and thus in potential Al-binding sites. The effect of these differences on cellular localisation as well as on tissue distribution of Al was compared in root tips of Zea mays and Vicia faba . The localisation of Al was assayed by Laser Microprobe Mass Analysis (LAMMA) after freeze-substitution. The radial mobility of Al was much lower in the roots of Vicia faba . In root tips of Zea mays , Al had reached the stele already after 60 min while it was confined to the rhizodermis and outer cortex cells in Vicia faba , indicating a stronger binding of Al in the cell walls of the dicoe. This binding, however, had no influence on intracellular distribution. Within 60 min intracellular Al was detectable in both species. Nevertheless, by far the highest Al concentrations were always measured in the cell wall.

On the distribution and transport of mineral elements in xylem, cambium and phloem of spruce (Picea abies [L.] Karst.)

The distribution of calcium, magnesium and potassium in the cambial region of a 20-year-old Norway spruce was determined by optical emission spectrometry (ICP-OES) in tissue samples ranging from the outer bark up to latewood and earlywood of the outer tree-rings. The highest contents of magnesium and potassium were found in the cambium and, in the case of calcium, in the developing phloem. In more detail we investigated the distribution and incorporation of calcium, magnesium and potassium in a model system of intact two-year-old plants (Picea abies [L.] Karst.). The roots of the seedlings, grown under controlled conditions, were removed from the soil and exposed to labelling solutions containing the enriched stable isotopes 25 Mg, 41 K, and 44 Ca as tracers. After seven days of labelling the cell walls of earlywood, latewood, cambium and phloem were analysed in stem cross sections of shock-frozen, cryo-substituted tissue by three microprobes: (i) by energy dispersive x-ray analysis (EDXA, 0.3 μm lateral resolution), (ii) isotope specific point analysis, using the laser microprobe mass analyser (LAMMA, 1.5μm lat. res.) and (iii) isotope specific imaging by secondary ion mass spectrometry (SIMS, 1-3μm lat. res.). After seven days of labelling approximately 60-75% of the Ca-content, 5-65% of the Mg-content and about 25-30% of the K-content within the cell walls of the shoot originated from the labelling solutions. The upper part (one-year-old) of the shoot axis contained a 10% smaller fraction of labelled Ca and a 10% larger fraction of labelled Mg in comparison to the more basal regions of the shoot (two-year-old). For all three elements we could not detect any significant radial gradient of the labelled fraction while comparing earlywood, latewood, cambium and phloem. We interpret our results as evidence for (i) a relevant bidirectional longitudinal transport and (ii) a radial element exchange between xylem, cambium and phloem, completed within the seven days of labelling.

Arbuscular mycorrhizas in phosphate-polluted soil: interrelations between root colonization and nitrogen

To investigate whether arbuscular mycorrhizal fungi (AMF) – abundant in a phosphate-polluted but nitrogen-poor field site – improve plant N nutrition, we carried out a two-factorial experiment, including N fertilization and fungicide treatment. Percentage of root length colonized (% RLC) by AMF and tissue element concentrations were determined for four resident plant species. Furthermore, soil nutrient levels and N effects on aboveground biomass of individual species were measured. Nitrogen fertilization lowered % RLC by AMF of Artemisia vulgaris L., Picris hieracioides L. and Poa compressa L., but not of Bromus japonicus Thunb. This – together with positive N addition effects on N status, N:P-ratio and aboveground biomass of most species – suggested that plants are mycorrhizal because of N deficiency. Fungicide treatment, which reduced % RLC in all species, resulted in lower N concentrations in A. vulgaris and P. hieracioides, a higher N concentration in P. compressa, and did not consistently affect N status of B. japonicus. Evidently, AMF had an influence on the N nutrition of plants in this P-rich soil; however – potentially due to differences in their mycorrhizal responsiveness – not all species seemed to benefit from a mycorrhiza-mediated N uptake and accordingly, N distribution.

Root cooling strongly affects diel leaf growth dynamics, water and carbohydrate relations in Ricinus communis.

In laboratory and greenhouse experiments with potted plants, shoots and roots are exposed to temperature regimes throughout a 24 h (diel) cycle that can differ strongly from the regime under which these plants have evolved. In the field, roots are often exposed to lower temperatures than shoots. When the root-zone temperature in Ricinus communis was decreased below a threshold value, leaf growth occurred preferentially at night and was strongly inhibited during the day. Overall, leaf expansion, shoot biomass growth, root elongation and ramification decreased rapidly, carbon fluxes from shoot to root were diminished and carbohydrate contents of both root and shoot increased. Further, transpiration rate was not affected, yet hydrostatic tensions in shoot xylem increased. When root temperature was increased again, xylem tension reduced, leaf growth recovered rapidly, carbon fluxes from shoot to root increased, and carbohydrate pools were depleted. We hypothesize that the decreased uptake of water in cool roots diminishes the growth potential of the entire plant - especially diurnally, when the growing leaf loses water via transpiration. As a consequence, leaf growth and metabolite concentrations can vary enormously, depending on root-zone temperature and its heterogeneity inside pots.

Fluorescence microtomography: external mapping of elements inside biological samples

X-ray fluorescence element micro tomography allows to determine the element specific inner structure of a sample with resolutions in the micron range. It has a wide range of applications in many disciplines and is ideally suited for investigating element distributions inside of biological bulk samples at a cellular level with minimal sample preparation. The high intensity hard x-ray microbeam required for this scanning technique is produced using parabolic compound refractive lenses at a third generation undulator source. The sample is scanned through the microbeam in both translation and rotation and the fluorescence radiation created in the sample is recorded by an energy dispersive detector. From this data, the element distribution on a virtual section through the sample is recovered by tomographic techniques. The excitation of the fluorescence by monochromatic x-rays yields a high signal to background ratio and a low detection limit. As an example, we have investigated the distribution of physiologically relevant ions on a virtual section through a freeze dried root of the mahogany plant. Absorption of the fluorescence radiation inside the sample has to be taken into account in tomographic reconstruction and ultimately limits the size of the sample that can be investigated. A self-consistent reconstruction technique not requiring the explicit knowledge of the absorption inside the sample has been developed. Further developments of the technique are discussed.

High-resolution element mapping inside biological samples using fluorescence microtomography

Sample preparation for element analysis of many biological tissues is difficult to achieve and prone to introduce contamination. Using x-ray fluorescence element microtomography (XFEMT) the element distribution on a virtual section across the sample can be determined with a resolution in the micrometer range. Fluorescence microtomograms of two plant samples are shown, demonstrating the possibility to map physiologically relevant ions, trace elements, and heavy metal pollutants at the cellular level. Attenuation effects inside the plant are corrected by a self-consistent tomographic reconstruction technique.