Genome Engineering for Crop Improvement. Группа авторов

in grain at levels that pose a health risk. Due to good sensitivity, it is especially suitable to image distribution of trace mineral nutrients like selenium (Se) or hazardous elements like arsenic (As) (Moore et al. 2010). Therefore, Nano SIMS was used to determine the localization of Se in wheat and As in rice. Combined synchrotron X‐ray fluorescence (S‐XRF) and Nano SIMS analysis utilized the strengths of both techniques. Selenium was concentrated in the protein surrounding the starch granules in the starchy endosperm cells and more homogeneously distributed in the aleurone cells but with Se‐rich hotspots. Arsenic was concentrated in the sub‐aleurone endosperm cells associated with the protein matrix, rather than in the aleurone cells. Nano SIMS indicated that the high intensity of As, identified in the S‐XRF image was localized in micrometer‐sized hotspots near the ovular vascular trace and nucellar projection.

      In addition, Nano SIMS was also used to localize Ag in rice grains. The images revealed that the silver is concentrated in the aleuronic layer of the rice bran. Its concentration decreases in the sub‐aleurone and becomes negligible in the endosperm. Accumulation of silver does not alter the grain morphology and chemical characteristics. The metal may be extracted from the bran after milling of the rice, thereby causing no hazards in associated foodstuffs (Sen Gupta et al. 2017).

2.3.5 Sample Preparation

      Sample preparation is a highly demanding but crucial step that is directly related to the quality and accuracy of imaging results. Though state‐of‐the‐art imaging tools are available today, the process is still severely limited by slow and demanding sample preparation protocols. In order to keep the anatomy and biochemistry of the cellular components as undisturbed as possible, it is essential that all biochemical and proteolytic processes are inactivated and the structures are immobilized and fixed in space and time by a “fixation step.” Two approaches are normally used to “fix” biological samples: a chemical and a physical one (Dong et al. 2016b). Though chemical fixation is the most common approach for specimen preservation in light and electron microscopy, it is not suitable for spectroscopic analysis where it is essential to obtain the anatomy to be able to retrieve quantitative spatial biochemical information (Perrin et al. 2015). Since chemical fixation can lead to artifacts such as contamination, leaching of mobile elements and modification of biomolecules, cryofixation is accepted as the only reliable fixation method for spectroscopic analysis (Schneider et al. 2002; Vogel‐Mikuš et al. 2014; Perrin et al. 2015; Dong et al. 2016b). The samples must be frozen in such a way that the growth of ice crystals is prevented in a process called vitrification (Scheloske et al. 2004). The current cryofixation workflows for the preparation of plant samples for imaging the elemental distribution (e.g. micro‐PIXE) (Vogel‐Mikuš et al. 2014) include manual cutting of plant material into small pieces, embedding of the pieces in a freezing medium (OCT), rapid freezing by immersing the sample in a cryogen cooled with liquid nitrogen (e.g. isopentane, propane), cryotome sectioning and freeze drying. Due to their hardness, grains can be imbibed (soaked for two to four hours, or more for very hard seeds, at 4 °C) and hand cut with a sharp razor blade to 100–200 μm thick slices, especially when analyzing the samples with MS‐based and FTIR techniques, where OCT could contaminate sections or can interact with the sample composition. Carboxymethylcellulose (CMC), gelatine and their combinations have been successfully used as media compatible with biomolecular imaging (Dong et al. 2016b).

After cutting, freeze‐drying is performed to obtain vacuum‐compatible samples (micro‐PIXE, micro‐MeV‐SIMS) and samples with low water content, which are required in micro‐FTIR. During dehydration, sample shrinkage is often observed, which usually results in an uneven surface, affecting surface spectroscopic techniques such as micro‐FTIR, micro‐Raman and micro‐MeV‐SIMS. Shrinkage can also lead to a mismatch between biochemical and anatomical features (e.g. when plant protoplasts shrink and adhere to the cell wall, it is impossible to distinguish whether the biochemical information originates from the cell wall or from the inner parts of the cell), making biological interpretation extremely difficult (Dong et al. 2016b).

2.4 Prospect

      The revolution in omics technology, particularly in metabolomics, has been closely linked to technological developments in mass spectrometry, currently the most efficient technology for characterizing molecular structures, and has led to significant progress in providing a comprehensive understanding of biological functions. Spatial information in mass spectrometric analysis can be preserved if the extraction process is avoided or limited to very small areas of the sample, which is then analyzed using new ionization techniques, such as MALDI and MeV or keV SIMS. In these techniques, the analytes are ionized and desorbed directly from the surface, thus avoiding traditional liquid extractions. They allow the sample to be scanned with the ion beam and the mass spectrum to be recorded at any point, resulting in the exact location in the sample where a particular compound was detected. By automating the process, images can be generated that show the spatial distributions of all compounds detected.

      Development of accelerator‐based nuclear microprobes (micro‐PIXE), the third and fourth generation of synchro tron facilities with micro‐and nano‐focused beams (SR‐micro‐XRF), LA‐ICPMS and Nano SIMShaveadvanced2D imaging of the elemental distribution in plants down to the subcellular level. Each of these techniques has its advantages and limitations, but they all provide a means to complement biomolecular imaging with elemental distributions. Inevitably, suitable sample preparation is a crucial step in the application of the existing up‐to‐date imaging techniques.

      Cereals and pseudo‐cereal plants that produce starchy grains are part of almost every meal we eat. Cereals are usually processed before consumption and certain nutritious compounds stored in the husk or embryonic tissue may be lost. Therefore, there is an urgent need to further explore these research tools that will provide better insights into the 2D and 3D spatial distribution of nutrients and minerals in cereals, and the mechanisms behind these distribution patterns.

Acknowledgement

      The work was supported by Slovenian Research Agency (ARRS) project no. J7‐9418, N1‐0105, N1‐0090, L4‐9305, J7‐9398 and N7‐0077, and program group Plant Biology (P1‐0212). Synchrotron Light Research Institute, Thailand is acknowledged for provision of beamtime (proposal number 3457) and Jitrin Chaiprapa for assistance with the measurements. Synchrotron Elettra, Italy and EU are acknowledged for provision of beamtime (proposal number 2008223 and 2008203) and Diane Eichert and Lisa Vaccari for assistance with the measurements.

References

      1 Bjarnholt, N., Li, B., D'Alvise, J., and Janfelt, C. (2014). Mass spectrometry imaging of plant metabolites – principles and possibilities. Nat. Prod. Rep. 31: 818–837. https://doi.org/10.1039/c3np70100j.

      2 Bonafaccia, G., Marocchini, M., and Kreft, I. (2003). Composition and technological properties of the flour and bran from common and tartary buckwheat. Food Chem. 80: 9–15. https://doi.org/10.1016/S0308‐8146(02)00228‐5.

      3 Boughton, B.A., Thinagaran, D., Sarabia, D. et al. (2016). Mass spectrometry imaging for plant biology: a review. Phytochem. Rev. 15: 445–488. https://doi.org/10.1007/s11101‐015‐9440‐2.

      4 Briggs, D. (1983). Analysis of polymer surfaces by SIMS, 3—preliminary results from molecular imaging and microanalysis experiments. Surf. Interface Anal. 5: 113–118. https://doi.org/10.1002/sia.740050307.

      5 Cheah, Z.X., Kopittke, P.M., Harper, S.M. et al. (2019). In situ analyses of inorganic nutrient distribution in sweetcorn and maize kernels using synchrotron‐based x‐ray fluorescence microscopy. Ann. Bot. 123: 543–556. https://doi.org/10.1093/aob/mcy189.

      6 Collings,