Michael R. Thorpe

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.

Jasmonic acid treatment to part of the root system is consistent with simulated leaf herbivory, diverting recently assimilated carbon towards untreated roots within an hour.

It is known that shoot application of jasmonic acid (JA) leads to an increased carbon export from leaves to stem and roots, and that root treatment with JA inhibits root growth. Using the radioisotope (11)C, we measured JA effects on carbon partitioning in sterile, split-root, barley plants. JA applied to one root half reduced carbon partitioning to the JA-treated tissue within minutes, whereas the untreated side showed a corresponding--but slower--increase. This response was not observed when instead of applying JA, the sink strength of one root half was reduced by cooling it: there was no enhanced partitioning to the untreated roots. The slower response in the JA-untreated roots, and the difference between the effect of JA and temperature, suggest that root JA treatment caused transduction of a signal from the treated roots to the shoot, leading to an increase in carbon allocation from the leaves to the untreated root tissue, as was indeed observed 10 min after the shoot application of JA. This supports the hypothesis that the response of some plant species to both leaf and root herbivores may be the diversion of resources to safer locations.

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.

Rapid cooling triggers forisome dispersion just before phloem transport stops

Phloem transport stops transiently within dicot stems that are cooled rapidly, but the cause remains unknown. Now it is known that (1) rapid cooling depolarizes cell membranes giving a transient increase in cytoplasmic Ca(2+), and (2) a rise of free calcium triggers dispersion of forisomes, which then occlude sieve elements (SEs) of fabacean plants. Therefore, we compared the effects of rapid chilling on SE electrophysiology, phloem transport and forisomes in Vicia faba. Forisomes dispersed after rapid cooling with a delay that was longer for slower cooling rates. Phloem transport stopped about 20 s after forisome dispersion, and then transport resumed and forisomes re-condensed within similar time frames. Transport interruption and forisome dispersion showed parallel behaviour--a cooling rate-dependent response, transience and desensitization. Chilling induced both a fast and a slow depolarization of SE membranes, the electrical signature suggesting strongly that the cause of forisome dispersion was the transient promotion of SE free calcium. This apparent block of SEs by dispersed forisomes may be assisted by other Ca(2+)-dependent sealing proteins that are present in all dicots.

AtSUC2 has a role for sucrose retrieval along the phloem pathway: evidence from carbon-11 tracer studies.

The location of the phloem within a plant, and its vulnerability to disruption, make it a difficult tissue to study and therefore non-invasive studies of phloem functionality are important. Here we compare, phloem transport, measured non-invasively, in wild type Arabidopsis thaliana, and transposon-insertion mutants for AtSUC1 or AtSUC2, giving in vivo information on the importance of these sucrose transporters for phloem transport. The suc2 mutant showed an increase in both phloem leakage and transport time, consistent with reduced sucrose uptake into both transport and collection phloem. The results are consistent with the AtSUC2 transporter being important for retrieval of leaked sucrose in the transport phloem of Arabidopsis. There was no difference in phloem transport properties between the wild type and the suc1 mutants, implying that the AtSUC1 transporter does not play a significant role within the transport phloem of Arabidopsis under the conditions of our study.

Pseudomonas fluorescens CHA0 maintains carbon delivery to Fusarium graminearum-infected roots and prevents reduction in biomass of barley shoots through systemic interactions

Soil bacteria such as pseudomonads may reduce pathogen pressure for plants, both by activating plant defence mechanisms and by inhibiting pathogens directly due to the production of antibiotics. These effects are hard to distinguish under field conditions, impairing estimations of their relative contributions to plant health. A split-root system was set up with barley to quantify systemic and local effects of pre-inoculation with Pseudomonas fluorescens on the subsequent infection process by the fungal pathogen Fusarium graminearum. One root half was inoculated with F. graminearum in combination with P. fluorescens strain CHA0 or its isogenic antibiotic-deficient mutant CHA19. Bacteria were inoculated either together with the fungal pathogen or in separate halves of the root system to separate local and systemic effects. The short-term plant response to fungal infection was followed by using the short-lived isotopic tracer 11CO2 to track the delivery of recent photoassimilates to each root half. In the absence of bacteria, fungal infection diverted carbon from the shoot to healthy roots, rather than to infected roots, although the overall partitioning from the shoot to the entire root system was not modified. Both local and systemic pre-inoculation with P. fluorescens CHA0 prevented the diversion of carbon as well as preventing a reduction in plant biomass in response to F. graminearum infection, whereas the non-antibiotic-producing mutant CHA19 lacked this ability. The results suggest that the activation of plant defences is a central feature of biocontrol bacteria which may even surpass the effects of direct pathogen inhibition.

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.

Modelling phloem transport within a pruned dwarf bean: a 2-source-3-sink system

A mechanistic model of carbon partitioning, based on the Münch hypothesis of phloem transport and implemented with PIAF-Münch modelling platform (Lacointe and Minchin 2008), was tested for an architecture more complex than any tested previously. Using 11C to label photosynthate, responses in transport of photosynthate within a heavily pruned dwarf bean plant (Phaseolus vulgaris L.) to changes in source and sink activities were compared with model predictions. The observed treatment responses were successfully predicted. However, the observations could not be completely explained if the modelled stem contained only one phloem pathway: tracer from a labelled leaf was always detected in both shoot apex and root, whichever of the two leaves was labelled. This shows that bidirectional flow occurred within the stem, with solute moving simultaneously in both directions. Nevertheless, a model architecture with very little more complexity could incorporate such bidirectional flow. We concluded that the model could explain the observations, and that the PIAF-Münch model platform can be expected to describe partitioning in even more complex architectures.

Hydrodynamics of steady state phloem transport with radial leakage of solute.

Long-distance phloem transport occurs under a pressure gradient generated by the osmotic exchange of water associated with solute exchange in source and sink regions. But these exchanges also occur along the pathway, and yet their physiological role has almost been ignored in mathematical models of phloem transport. Here we present a steady state model for transport phloem which allows solute leakage, based on the Navier-Stokes and convection-diffusion equations which describe fluid motion rigorously. Sieve tube membrane permeability Ps for passive solute exchange (and correspondingly, membrane reflection coefficient) influenced model results strongly, and had to lie in the bottom range of the values reported for plant cells for the results to be realistic. This smaller permeability reflects the efficient specialization of sieve tube elements, minimizing any diffusive solute loss favored by the large concentration difference across the sieve tube membrane. We also found there can be a specific reflection coefficient for which pressure profiles and sap velocities can both be similar to those predicted by the Hagen-Poiseuille equation for a completely impermeable tube.

Leaf photosynthetic and solar-tracking responses of mallow, Malva parviflora, to photon flux density

Malva parviflora L. (mallow) is a species that occupies high-light habitats as a weedy invader in orchards and vineyards. Species of the Malvaceae are known to solar track and anecdotal evidence suggests this species may also. How M. parviflora responds physiologically to light in comparison with other species within the Malvaceae remains unknown. Tracking and photosynthetic responses to photon flux density (PFD) were evaluated on plants grown in greenhouse conditions. Tracking ability was assessed in the growth conditions and by exposing leaves to specific light intensities and measuring changes in the angle of the leaf plane. Light responses were also determined by photosynthesis and chlorophyll fluorescence. Leaves followed a heliotropic response which was highly PFD-dependent, with tracking rates increasing in a curvilinear pattern. Maximum tracking rates were up to 20 degrees h(-1) and saturated for light above 1,300 micromol (photons) m(-2) s(-1). This high-light saturation, both for tracking (much higher than the other species), and for photosynthesis, confirmed mallow as a high-light demanding species. Further, because there was no photoinhibition, the leaves could capture the potential of an increased carbon gain in higher irradiance by resorting to solar tracking. Modelling suggested the tracking response could increase the annual carbon gain by as much as 25% compared with leaves that do not track the sun. The various leaf attributes associated with solar tracking, therefore, help to account for the success of this species as a weed in many locations worldwide.