Root type contribution to phosphate nutrition of rice during asymbiosis and interaction with symbiotic fungi.

Phosphate (Pi) is an essential plant macronutrient and occurs in scarce amounts in most soils, which frequently limits plant growth. Plant fitness and crop productivity is directly influenced by the plant root system's efficiency in exploring soil nutrient resources. Plant root systems are composed of distinct root types (RTs), and their ratio and spatial arrangement define root system architecture and nutrient foraging efficiency. RTs may individually respond to their abiotic and biotic soil microenvironment, thereby asymmetrically contributing to plant nutrient acquisition. Consistently, there is a growing interest of scientist and breeders in classifying and quantifying root system architectural traits (reviewed in Rogers and Benfey, 2015) towards 'designer crops with optimal root system architecture for nutrient acquisition' (Kong et al., 2014). Surprisingly however, RTs are 'under-investigated' in particular in adult crops where RT-focused research is missing. The Paszkowski group has addressed this gap in knowledge by studying the RTs of adult rice plants at V8 stage (before flowering). The root system of adult rice is composed of three RTs, the crown, large and fine lateral roots (CR, LLR, FLR, respectively) with distinct developmental and anatomical characteristics. Unique transcriptional signatures for each rice RT were revealed, which suggested distinct roles in nutrient acquisition (Gutjahr et al., 2015). Plants acquire Pi either directly or in association with naturally prevalent arbuscular mycorrhizal (AM) fungi. In rice, the different RTs exhibit variable extent of colonization with LLR fully and CR partially colonized, whereas FLRs remain non-colonized (Gutjahr et al., 2009). Exposing rice roots to a beneficial AM fungus led to the profound modulation of each RT transcriptome, indicative of a switch in their functional relationship. The contribution of individual RTs to rice Pi nutrition in asymbiosis or during interaction with AM fungi remains at present unclear and represents the main objective of this proposal. In addition, the Paszkowski group has recently discovered that exclusively vacuoles of fungus-containing plant cells accumulated structures resembling polyphosphate-storage bodies (Roth & Paszkowski, unpublished), previously not described for higher plants. Colonized and non-colonized RTs thus appear to differ in the spatial distribution and speciation of tissue phosphorus, which may impact on the nutritional physiology of the plant. The proposed work aims at quantitatively and spatially determining Pi uptake and partitioning into tissue and cellular pools across the RTs of adult rice root systems. The application of a unique combination of interdisciplinary techniques in analytics, imaging and molecular genetics will deliver an insight into in situ phosphorus fluxes at unprecedented spatio-temporal resolution. As a staple food for more than half of the human population, rice is central for food security. The study will inform about RT functioning, important for rational breeding approaches towards improved plant stress tolerance and crop productivity.

This project uses state of the art instrumentation and techniques to provide a high resolution map of phosphate uptake and tissue distribution across individual root-types of the rice root system. This project is unique due to (a) its focus on rice root-types, the building units of the root system, and (b) due to an unparalleled combination of techniques. Compartmentalized plant growth containers enable the root-type specific measurement of radiolabeled phosphate uptake rates (I Jakobsen, Denmark). In parallel, the involvement of either the direct or the symbiotic phosphate uptake pathway, and tissue phosphorus fluxes will be documented by live imaging of fluorescent rice marker lines using the latest generation multiphoton confocal microscope (J Skepper), which enables deeper tissue imaging.
The above described methodologies will be linked with measuring tissue phosphorus concentrations from PPM to hundreds of mmol/L and across overlapping scales of spatial resolution from millimetres to sub-micrometre. The global phosphorus tissue content of each root-type is determined by ICP-MS to the scale of PPM (D Salt). 31P-NMR (M Duer, D Reid) and XRF/ XANES (K Ignatyev) will provide detailed information of the distribution and concentrations of inorganic and organic phosphorus. Energy dispersive x-ray microanalysis of cryo-immobilised cross or longitudinally cryo-planed roots provides sub nanometre resolution and quantification of phosphorus in the range of 1 - 200 mmol/L in volumes of < 0.5 micron3 (J Skepper).
All techniques selected for this approach have previously been individually established on plant material. Therefore, the technical feasibility of the projected work is high. A novel, more profound and comprehensive understanding is generated by the combination of these overlapping and carefully selected techniques, which will give a superlative description of phosphorus dynamics and quantities by in vivo and ex vivo method of imaging and quantitative analyses.

The project will achieve academic, economic and social impacts.

Academic impact will be realized in diverse areas of plant biology: (a) In nutritional plant physiology as our approach focuses on the macronutrient phosphate and its spatio-temporal acquisition by the modules of the rice root system, the root-types, in asymbiosis and during interaction with beneficial fungi. (b) The attention of cell biologists is attracted by integrating cell biology with whole plant physiology via providing an insight into sub-cellular phosphorus concentrations and speciation across decreasing magnification scales to whole root-type tissue. (c) The functional dissection of phosphate uptake pathways will be of importance to molecular biologists and geneticists interested in the molecular mechanisms underlying regulation of nutrient acquisition. (d) Root developmental biology is an intense research field, typically concentrating on roots of plants at seedling stage. The root types of the adult rice root system have distinct developmental characteristics with functional implications for nutrient uptake that have molecularly been rarely investigated.
The application of comprehensive and interdisciplinary methodologies including imaging, chemical analytics as well as genetics and physiology to a biological question as addressed here is unique. Therefore academic impact will in addition be achieved through pioneering an innovative way of working that will provide insights into nutrient fluxes in the cereal crop rice. It will furthermore impact on the experimental routines towards the combined utilization of otherwise independent methodologies.

The importance of root system architectural traits for crop productivity has long been recognized and breeding programs have been designed accordingly. As this project will provide information on the root-type specific contribution to rice phosphate nutrition, economic impact will be achieved through the implementation of the generated knowledge in breeding programs, leading to increased crop productivity. Rice is a staple food for more than half of the human population and is therefore central for food security plus a target for the design of sustainable strategies to increase crop yield. Findings made in rice can immediately be translated from fundamental to applied research without the need to transfer knowledge across phylogenetically distant plant species. Moreover, rice represents the primary model organism for cereal crops, frequently permitting the extrapolation of discoveries to other cereals such as wheat, barley and maize.

Societal impact of the proposed work will be achieved through stimulating the public's attention to research and issues related to plant biology, in particular to crop science. Our project can serve as an example case to illustrate in a public-friendly fashion the power of linking fundamental biological questions with modern technologies and food security. We intend to initiate public demonstrations of the project to publicise our work via engaging in presentations within schools and at events such as the annual Cambridge Science Festival.