Arabidopsis thaliana is the most widely used model organism for research in plant biology. While significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of Arabidopsis, extracting and understanding the functional framework of metabolism is challenging, both from a technical perspective due to losses and modification during extraction of metabolites from the leaves, and from the biological perspective, due to random variation obscuring how well the function is performed. The purpose of this work is to establish the in vivo metabolic profile directly from the Arabidopsis thaliana leaves without metabolite extraction, to reduce the complexity of the results by multivariate analysis, and to unravel the mitigation of cellular complexity by predominant functional periodicity. To achieve this, we use the circadian cycle that strongly influences metabolic and physiological processes and exerts control over the photosynthetic machinery. High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to obtain the metabolic profile directly from intact Arabidopsis leaves. Combining one- and two-dimensional 1H HR-MAS NMR allowed the identification of several metabolites including sugars and amino acids in intact leaves. Multivariate analysis on HR-MAS NMR spectra of leaves throughout the circadian cycle revealed modules of primary metabolites with significant and consistent variations of their molecular components at different time points of the circadian cycle. Since robust photosynthetic performance in plants relies on the functional periodicity of the circadian rhythm, our results show that HR-MAS NMR promises to be an important non-invasive method that can be used for metabolomics of the Arabidopsis thaliana mutants with altered physiology and photosynthetic efficiency.

Interactions between plants and the soil's microbial & fungal flora are crucial for the health of soil ecosystems and food production. Microbe-plant interactions are difficult to investigate in situ due to their intertwined relationship involving morphology and metabolism. Here, we describe an approach to overcome this challenge by elucidating morphology and the metabolic profile of Medicago truncatula root nodules using Magnetic Resonance (MR) Microscopy, at the highest magnetic field strength (22.3 T) currently available for imaging. A home-built solenoid RF coil with an inner diameter of 1.5 mm was used to study individual root nodules. A 3D imaging sequence with an isotropic resolution of (7 μm) 3 was able to resolve individual cells, and distinguish between cells infected with rhizobia and uninfected cells. Furthermore, we studied the metabolic profile of cells in different sections of the root nodule using localised MR spectroscopy and showed that several metabolites, including betaine, asparagine/ aspartate and choline, have different concentrations across nodule zones. The metabolite spatial distribution was visualised using chemical shift imaging. Finally, we describe the technical challenges and outlook towards future in vivo MR microscopy of nodules and the plant root system. Interactions between plants and microbes are considered to be important for the health of the soil ecosystem as a whole 1. Understanding the metabolic interactions between plants and microbes, both commensal and parasitic, could help address many of the challenges we face today, related to agriculture and food security. One such interaction is the microbiome-mediated uptake of nitrogen by plants. Availability of biologically-active forms of nitrogen in the soil is an important factor determining crop yield. Current agricultural practice, therefore, relies strongly on nitrogen fertiliser to supplement soil nitrogen to ensure high crop yield 2. The Haber-Bosch nitrogen fixation process, used to produce the ammonia needed for these fertilisers, currently consumes 1% of the world energy sources; making it the most energy consuming process in the chemical industry 3. In contrast, alternative processes (e.g. symbiotic nitrogen fixation, SNF) achieve the same result of fixing nitrogen without the need for high pressure and temperature required by the Haber-Bosch process. More precisely, plants have solved the problem of biological nitrogen fixation through commensal processes, involving bacterial infection of plant roots 4. Mutualistic infections are omnipresent in nature, with a wide range of nitrogen-fixing bacteria-such as those that are collectively named rhizobium-invading not just plants but also the phycosphere of green algae 5. The mutualistic symbiosis involves almost all parts of the plant cell machinery, including plastids and mitochondria. Of particular interest are the interactions between rhizobial bacteria and leguminous plants, which form dedicated organs-root nodules-to accommodate the bacteria. Medicago.

Botryococcus braunii is an oleaginous green algae with the distinctive property of accumulating high quantities of hydrocarbons per dry weight in its colonies. Large variation in colony structure exists, yet its implications and influence of oil distribution and diffusion dynamics are not known and could not be answered due to lack of suitable in vivo methods. This publication seeks to further the understanding on oil dynamics, by investigating naturally relevant large (700–1500μm) and extra-large (1500–2500μm) sized colonies of Botryococcus braunii (race B, strain Showa) in vivo, using a comprehensive approach of chemical shift selective imaging, chemical shift imaging and spin echo diffusion measurements at high magnetic field (17.6T). Hydrocarbon distribution in large colonies was found to be localised in two concentric oil layers with different thickness and concentration. Extra-large colonies were highly unstructured and oil was spread throughout colonies, but with large local variations. Interestingly, fluid channels were observed in extra-large colonies. Diffusion-weighted MRI revealed a strong correlation between colony heterogeneity, oil distribution, and diffusion dynamics in different parts of Botryococcus colonies. Differences between large and extra-large colonies were characterised by using T2 weighted MRI along with relaxation measurements. Our result, therefore, provides first non-invasive MRI means to obtain spatial information on oil distribution and diffusion dynamics in Botryococcus braunii colonies.

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This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RFfield homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 μm 3 , 2.2 pL) 3D imaging of a root sample.