Genome Engineering for Crop Improvement. Группа авторов

as shown in the microscopic image (a) and corresponding maps related to esters, representing fats (b), amide II representing proteins (c) and coupling of the CO and CC stretching representing starch (d). SR‐FTIR spectra were recorded on the SISSI beamline, Elettra, Trieste, Italy using a global source and a focal plane array detector. The maps represent 64 × 64 pixels with each pixel including 128 scans. Spectra were processed and maps were generated in Opus software (Bruker, USA).

2.3 Exploring the Mineral Distribution in Grain

      Development of accelerator‐based nuclear microprobes (micro‐Particle Induced X‐ray Emission; PIXE) (Moretto 1996), the third and fourth generation of synchrotron facilities with micro‐and nano‐focused beams (SR‐micro‐X‐ray Fluorescence (XRF) (Kaulich et al. 2009; Tolrà et al. 2011; Martínez‐Criado et al. 2016; Cotte et al. 2017) and Laser Ablation‐Inductively Coupled Mass Spectrometry (LA‐ICPMS) (Limbeck et al. 2015) have advanced the imaging of the elemental distribution in plants down to the subcellular level (Koren et al. 2013). Each of these techniques has its advantages and limitations, but provide a means to complement biomolecular imaging with elemental distributions.

2.3.1 Nuclear Microprobe

      With the nuclear microprobe or micro‐PIXE it is possible to image a broad spectrum of elements (from NatoU) with lateral resolution down to the submicron range (Vavpetič et al. 2017), but the method is less sensitive for light and heavy elements, with a detection limit in the μg g⁻¹ range for middle Z elements (Nečemer et al. 2008). Microprobes are normally operated in vacuum, therefore samples must be vacuum compatible – dehydrated or measured under cryogenic conditions (Simičič et al. 2002; Vogel‐Mikuš et al. 2014).

      The localization of minerals in whole grain cross sections of wheat was first performed in 1981 and 1985 with micro‐PIXE by Mazzolini, Pallaghy and Legge (Mazzolini et al. 1981, 1985), with special emphasis on the Zn and Mn distribution. It was found that both elements are mainly distributed in the aleurone layer and in the embryo. However, in the embryo, the highest Zn concentration was measured in the scutellum and the highest Mn concentration in the coleoptyle. Almost 30 years later, the same question of Zn allocation in the wheat grain, after Zn fertilization, was addressed and confirmed that Zn is preferentially localized in scutellum and aleurone (Pongrac et al. 2013a). With Zn fertilization (Zn added as ZnNO₃), the Zn concentrations in the aforementioned parts increased. In addition, almost twice as high Zn concentrations were also found in the endosperm, the part used for flour production. These results underline agronomic biofortification, i.e. increasing the mineral element composition of staple foods through the application of fertilizers, as a means to successfully increase the Zn concentration in the edible parts of the grain, and demonstrate the applicability of imaging techniques such as micro‐PIXE to evaluate the success of biofortification. Recently, grain cross‐section micro‐PIXE analysis of contrasting lines of barley (Hordeum vulgare L.) that accumulate different total amounts of Zn, additionally confirmed that differences in grain Zn accumulation apply to all parts of the grain, including the endosperm (Detterbeck et al. 2016), while also highlighting the possibility of selecting high Zn content lines for biofortification purposes.

      According to the World Health Organization, WHO, iron (Fe) deficiency affects almost two thirds of the world population, especially women in their fertility period and children (Collings et al. 2013). In wheat grains, Fe is found in the outer layers of bran, most of which is lost during milling and processing (Zhang et al. 2010; Regvar et al. 2011). The flour is almost free of Fe. Therefore, the development of wheat varieties with Fe‐enriched endosperm offers an important strategy to improve the nutritional availability of Fe in wheat flour. In a study conducted by Singh et al. (Singh et al. 2013, 2014), the variability of the mineral distribution in wheat was investigated by comparing three wheat genotypes and a wild wheat relative, Aegilops kotschyi (A.kot), which differ in the content of Fe in whole grain. In the genotypes with high Fe content (IITR26, A.kot), an increased concentration of Fe was observed in the aleurone layer, but also in the endosperm (Singh et al. 2014).

Beans are staple foods in many countries worldwide, especially in South and Central America. Thirteen genotypes of Phraseoulus vulgaris, P. coccineus and P. lunatus were investigated for Fe distribution (Cvitanich et al. 2010). It was shown that in P. vulgaris and P. coccineus high Fe concentrations are distributed in the cells surrounding the provascular tissue. Using the Pearls staining method, Fe was detected in the cytoplasm of epidermal cells of the cotyledons, in cells near the epidermis and in cells surrounding the provascular tissue. In contrast, the protein ferritin, which has been proposed as the most important Fe storage protein in legumes, was only detected in the amyloplasts of the seed cotyledons. With micro‐PIXE it was shown that the tissue near the provascular bundles contained up to 500 μg g⁻¹ Fe, depending on the genotype. In contrast to P. vulgaris and P. coccineus, P. lunatus did not show Fe accumulation in the cells surrounding the provascular tissues of the cotyledons. The results presented emphasize the importance of complementing research on model organisms with analysis in crops and suggest that Fe distribution criteria should be integrated into selection strategies for the Fe biofortification of beans (Cvitanich et al. 2010).

The application of micro‐PIXE has enabled us to generate quantitative element maps of the nutritionally most important elements in the grain tissue of common and Tartary buckwheat (Vogel‐Mikuš et al. 2009; Pongrac et al. 2011, 2013b, 2020). About half of the buckwheat grain (57% of common and 43% of Tatary buckwheat) consists of starchy endosperm, which is only a moderate source of minerals. This could be due to dilution effects, with starch granules being formed in the last stages of assimilation and filling of the endosperm cells, which are not as rich in elements as other grain components. However, the majority of elements, which are found in both buckwheat varieties, are localized in the cotyledons, while Ca is mainly localized in the seed coat (Vogel‐Mikuš et al. 2009; Pongrac et al. 2011, 2013b). Though the localization patterns are quite similar in both buckwheat species, higher contractions of Mg, P, Mn, Fe, Cu and Zn are observed in the cotyledons of Tartary buckwheat grain (Figure 2.5), making the latter a better source of minerals compared to common buckwheat grain. During the processing of buckwheat grains, the outer parts of the grains are removed (husk, seed coat and aleurone layer), while the inner parts with endosperm and cotyledons are preserved – the de‐husked product is called groats. In wheat, however, the flour‐milling fraction contains only parts of the endosperm, which is rather poor in minerals. A comparison between the edible grain fractions of buckwheat (endosperm and cotyledons) and three different wheat genotypes (endosperm) shows that buckwheat groats are a much richer source of minerals than wheat flour. However, the bioavailability of Fe, Zn, Mn, and Mg in buckwheat groats may be affected negatively because of the high P concentrations found in the groats (more precisely, in cotyledons). In grain, the majority of P is present in the form of phytate, which limits bioavailability of phytate‐bound essential elements such as Mg, Mn, Fe and Zn found in buckwheat cotyledons.

2.3.2 Synchrotron Radiation X‐Ray Florescence Spectrometry

SR‐micro‐XRF is a state‐of‐the‐art imaging technique that enables 2D and 3D imaging. Separate beamlines are usually dedicated to low or high energy X‐ray fluorescence. Low SR energy micro‐XRF is operated in vacuum (e.g. TwinMic, Synchrotron Elettra Trieste (Gianoncelli et al. 2016), ID21, ESRF, Grenoble (Cotte et al. 2017), while hard X‐ray microprobes are operated in air with limited sensitivity for low Z elements (Martínez‐Criado et al. 2016). The detection limits range down to below μg g⁻¹ and the lateral resolution to a few tens of nanometers (Martínez‐Criado et al. 2016). The highest chemical sensitivity can be achieved by tuning the photon beam energy above the absorption edge of the element of interest; however, this limits the number of elements that can be examined during the fingerprinting of the samples. In addition to SR‐micro‐XRF, a bench‐top laboratory XRF instrument with a focused beam and polychromatic excitation (e.g. Tornado, Bruker) can be used,