Genome Engineering for Crop Improvement. Группа авторов

the introduction of the new cluster and swift ion beams (MeVSIMS) (Nakata et al. 2008), the ion yield in the higher mass range has been significantly improved (Jeromel et al. 2014), allowing the detection of intact lipids and secondary metabolites (Jenčič et al. 2016, 2017). However, ion intensities in them/z ~ 800 range are still orders of magnitude lower than intensities around m/z ~ 100, and sensitivity to intact metabolites is thus significantly lower than with other MS imaging techniques. Due to the high degree of fragmentation in SIMS experiments, characteristic fragments of analytes are often imaged rather than the intact analytes. SIMS does not require much sample preparation (no matrix is needed as with MALDI), but the fact that the analysis takes place in a vacuum means that the samples need to be freeze‐dried before being introduced into the vacuum or measured under cryogenic conditions (Dickinson et al. 2006).

An example of MeV‐SIMS imaging of organic compounds is presented in Figure 2.1 obtained as described previously (Jeromel et al. 2014).

2.2.3 Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared (FTIR) imaging represents an important addition to the spatially resolved study of the molecular composition of plants. FTIR spectroscopy with a focused beam provides reliable information on biochemical composition (structural molecules) in a spatially resolved manner, without chemical fixation or staining of the sample, but plant tissue samples still need to be cryofixed and cutinto10 μm thin sections to perform micro‐FTIR measurements in the most common transmission mode (Regvar et al. 2013). An important advantage of FTIR over other imaging techniques is that infrared radiation does not cause radiation damage to biological material, so that the chemical nature of the sample constituents can be studied without breaking the chemical bonds (Miller and Dumas 2006). This enables subsequent complementary studies using other techniques on the same samples and areas of interest.

Figure 2.1 Spatial distribution of organic compound fitting the peak of m/z = 182, presumably belonging to phenolics in a wheat grain cross‐section imaged by MeV‐SIMS. Color bar presents the total ion yield of the selected peak.

      FTIR imaging can discriminate between individual cells in plant samples. Cell walls and large structures within cells such as starch granules and protein bodies, in intact hydrothermally processed and digested wheat (Triticum aestivum) kernels, were observed. Gelatinized and native starch within cells could be distinguished, and also the loss of starch during wheat digestion was observed (Warren et al. 2015).

In barley grain, FTIR imaging was used to visualize the spatial distribution of sucrose (Suc). The intake of Sucin barley caryopsis occurs via the main vascular bundle, which is located in the ventral pericarp. As a result, concentration gradients are formed along the allocation path, which are nicely represented in the Suc distribution maps obtained by FTIR. The concentration of Suc in the post‐phloem regions of the pericarp drops slightly and increases again in the region of the ETC. Such gradients could be an indication of passive unloading at the vein, but an active loading against the concentration gradient at the ETC via a set of sugar transporters. From the ETC, most of the Suc moves passively into the storage tissue, resulting in the formation of a steep negative gradient from the ETC toward the peripheral endosperm. Besides Suc, FTIR can also analyze the distribution of other metabolites such as starch. The quantitative data identified a characteristic pattern of starch distribution, which has already been described, and is largely explained by different local conditions that favor starch biosynthesis under hypoxic conditions in the endosperm (Guendel et al. 2018).

Of the edible grains, buckwheat is a pseudo‐cereal and it is one of the main traditional ancient food sources in some of the populations around the Himalayan region (southwest China, Bhutan, Nepal, northern India and northeast Pakistan). It is also a popular food in several other countries such as Slovenia, Poland, the Czech Republic, Ukraine, Korea, and Japan (Bonafaccia et al. 2003). In common (Fagopyrum esculentum) and Tartary (Fagopyrum tataricum) buckwheat grains, which produce gluten‐free starch‐rich grains, chemical profiling using a combination of ATR and synchrotron radiation FTIR revealed the distinct composition of each of the three main tissues (husk, cotyledons and endosperm) and illustrated their difference from the whole grain (Figure 2.2). Chemical mapping of a cross‐section of the Tartary buckwheat grain clearly illustrates two distinct grain tissues, the cotyledons and the endosperm (Figure 2.3). Likewise, in wheat grain, husk, aleurone and endosperm also exhibit distinct chemical profiles (Figure 2.4).

Figure 2.2 Comparison of the infrared spectra of common buckwheat (pink) and Tartary buckwheat (blue) whole grain and grain tissues endosperm, cotyledons and husk. The determination of the main molecular groups present in the entire grain was firstly assessed via the Attenuated Total Reflection (ATR) mode. Tissue‐specific spectra were collected from cryo‐sectioned and freeze‐dried grain and data was processed as described previously (Regvar et al. 2013). The recorded spectra were corrected for atmospheric changes, truncated to a range of 3800–800 cm⁻¹, baseline corrected and normalized to permit an accurate comparison of the different spectral domains and their intensities. Peak A, lipids (2990–2813 cm⁻¹); peak B, ester band (1767–1714 cm⁻¹); peak C, amide I (1700–1586 cm⁻¹); peak D, amide II (1573–1490 cm⁻¹); peak E, lignin (1530–1490 cm⁻¹); peak F, cellulose (1278–1219 cm⁻¹); peak G, total carbohydrates (1182–886 cm⁻¹); and peak H, carboxyl groups from ligands, proteins, various polysaccharides (1349–1308 cm⁻¹).

Figure 2.3 Representative SR‐FTIR chemical mapping of the Tartary buckwheat grain cross‐section comprising the cotyledon (c) and endosperm (e) as shown in the microscopic image (a) with the red rectangle indicating the area of mapping and the corresponding maps related to lipids (CH₂, b), lipids (CH₃, c), esters (d), amide I (e), amide II (f), total carbohydrates (g), cellulose (h) and lignin (i) SR‐FTIR spectra were recorded on the SISSI beamline, Elettra, Trieste, Italy as described previously (Regvar et al. 2013).

Figure 2.4 Representative