Spatial Distribution of Polyphenolic Compounds in Corn Grains (Zea mays L. var. Pioneer) Studied by Laser Confocal Microscopy and High-Resolution Mass Spectrometry

Desirable changes in the biochemical composition of food plants is a key outcome of breeding strategies. The subsequent localization of nutritional phytochemicals in plant tissues gives important information regarding the extent of their synthesis across a tissue. We performed a detailed metabolomic analysis of phytochemical substances of grains from Zea mays L. (var. Pioneer) by tandem mass spectrometry and localization by confocal microscopy. We found that anthocyanins are located mainly in the aleurone layer of the grain. High-performance liquid chromatography in combination with ion trap tandem mass spectrometry revealed the presence of 56 compounds, including 30 polyphenols. This method allows for effective and rapid analysis of anthocyanins by plotting their distribution in seeds and grains of different plants. This approach will permit a more efficient screening of phenotypic varieties during food plant breeding.


Introduction
The consumption of corn for 2018-2019 reached 315 million tons in the USA, 276 million tons in China, 63 million tons in the European Union, and 66 million tons in Brazil. In maize breeding, the discovery of genes responsible for the formation of corn endosperm accelerated research on the modeling of nutritional and taste properties of the corn.
The biochemical composition of corn grains, including protein, fatty acid, saccharide, and phenolic content, significantly affect the nutritional quality and taste of corn. The content of essential amino acids, such as valine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, phenylalanine, histidine, and arginine is one of the major factors that determine the nutritional value of corn [1].
Corn grains have the highest polyphenol content (6056.9 mg/kg dry weight or 15.55 µmol/g) among other grains and represent significant interest for phytochemical and metabolomic study [2,3]. Phenolic compounds can have radical scavenging, chelating and Figure 1. Structure of the grain of dent corn (with the symbolic designation of parts of the grain) (modified from [10]).
Previous research organized phenolic compounds according to the degree of antioxidant activity: simple phenolic acids < hydroxycinnamic acids < flavonols < flavan-3-ols < dimers of procyanidins [6]. It is known that the antioxidant activity of phenolic acids increases with an increase in the distance separating the carbonyl group and the aromatic ring, and hydroxycinnamic acid derivatives have stronger antioxidant activity than benzoic acid derivatives [11]. The 7,8-double bond of hydroxycinnamic acids also enhances their antioxidant potential, compared with hydroxybenzoic acids.
Jigh-performance liquid chromatography (HPLC) was predominantly used to identify carotenoids [12][13][14] and polyphenols [15,16] in corn grains. A review by Ranilla (2020) summarized the application of metabolomics for the characterization of metabolites in corn grains and emphasized the importance of phenotype-genotype studies aimed to explore corn genetic diversity [17]. The application of electrospray ionization mass spectrometry (ESI-MS) in combination with HPLC is a cost-effective and statistically robust method for high-throughput phenotypic characterization of corn [18]. The HPLC-ESI-MS/MS analytical configuration is widely used for the characterization of phenolic bioactive compounds in worldwide corn biodiversity. Montilla et al. (2011) characterized 10 corn landraces based on the content of phenolic fractions [19]. Das and Singh (2016) characterized four corn hybrids based on the content of phenolic acids, anthocyanins, and flavonols [20].
Another important problem is the study of spatial distribution and composition of phytochemicals in corn grains. Microscopic images are widely used as important sources of information on morphometric characteristics of cells and the architecture of plant tissue [21]. Confocal laser scanning microscopy was previously used to localize the phenolic compounds in different plants [22]. Morphological and biochemical changes in roots of corn Zea mays L. were previously studied by confocal microscopy [23,24]. To the best of the authors' knowledge, no published studies report an application of confocal microscopy for the identification of phytochemicals in the grains of corn Zea mays L.
Considering the qualitative data of phytochemical composition obtained by HPLC-MS and literature information regarding the optical properties of identified chemicals, the combination of HPLC data with fluorescence microscopy is a good opportunity to explore the localization of phenolic compounds in plants. The combination of these methods is important for breeding since it allows us to assess whether the genes involved in the synthesis of these substances are expressed only in certain tissues (e.g., the aleurone layer, the germ layer, the vitreous endosperm) or in all grain glutes uniformly. In addition, this approach makes it possible to estimate the number and size of storing organelles (granules, chloroplasts, vesicles), since selection is important in both increasing their number and increasing their size. Thus, the combination of these methods allows us to obtain more complex information about the studied plants.
In this study, we used combined mass spectrometry and confocal laser microscopy to determine the structural properties and phytochemical composition of corn grains. In our case, the combination of HPLC-MS and fluorescence microscopy allowed us to demonstrate the localization of polyphenolic compounds in the grains of corn Zea mays L. However, the interpretation of the results of this study requires taking into account the limitations of the study design. The application of combined HPLC and fluorescent microscopy includes the possibility of spatial localization of different groups of plant chemicals in general but not the individual compounds.

Tandem Mass Spectrometry
The extracts of corn grains were analyzed using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) to explore the diversity of available phytochemicals. The structural identification of each compound was carried out based on their accurate mass and MS/MS fragmentation using LC-ESI MS. In total, 56 compounds were successfully identified and characterized by comparing fragmentation patterns with those available in the literature. The results of a preliminary study showed the presence of 56 compounds corresponding to the genus Zea, some of which were identified for the first time in Zea mays L. The identified compounds, with molecular formulas m/z calculated and observed MS/MS data, and their comparative profile for corn grains are summarized in Table A1. The chromatograms of total compounds in the grain extract in positive and negative ionization modes are presented in Figure 2. In the present study, 30 polyphenol compounds were identified and characterized. In addition, 26 compounds of other classes were identified, including identified for the first time in corn grains oxylipins 13-trihydroxy-octadecenoic acid and 9,12,13-Trihydroxy-trans-10-octadecenoic acid.   [28,29], strawberry [30].  According to the literature data, strong blue fluorescence of plant grains under UV excitation could be explained by the presence of phenolic compounds such as hydroxycinnamic [31] or ferulic acid [32], and lignin [33]. The endosperm reveals very low blue autofluorescence (Figures 6 and 7) due to the very low amount of phenolic substances in the endosperm cells of seeds and grains [34]. It was reported that the pericarp of Zea mays had a total phenolic content 30-34 fold higher than endosperm [35]. Our results demonstrated that the aleurone cells (Figures 5b, 6b and 7b) and embryo (Figure 7b) were enriched with blue autofluorescence substances. At the same time, it is known that no lignin is present in aleurone [36], but hydroxycinnamic, ferulic, and coumaric acids were reported in aleurone cells of cereals [37,38]. Therefore, the observed blue fluorescence might be caused by hydroxycinnamic, ferulic, and coumaric acids. The main blue fluorescent compound in the pericarp is lignin, which is a heterogeneous mixture of randomly polymerized phenolic monolignols [39].

Confocal Microscopy
The emission in the red spectrum mainly occurs due to the presence of various polyphenolic compounds, including anthocyanins and anthocyanidins [40].

Discussion
It is known that polyphenols have strong antioxidation, anticancer, anti-infection, and other valuable activities [41]. The knowledge of polyphenol distribution in plants will benefit the development of the methods of their direct extraction and further application in the food, pharmaceutical, and cosmetic industries.
Another important problem is the influence of environmental conditions on the polyphenol composition of the plants. The significant genotypic effects and interactions of the genotype with the environment suggest that breeding methodology will require careful site selection and accounting for changes in genotype rank with changes in cultivation sites.
The important characteristics such as grain color, protein, and polyphenol distribution represent significant interest for breeding. In the grain images, the fluorescence signal under UV excitation (405 nm) comes from ferulic acid [42] and lignin [33]. It should be noted that lignin is absent in aleurone, while coumaric and diferulic acids are present in the walls of aleurone cells. These acids can contribute to the autofluorescence of these cell walls [43,44].
Autofluorescence in the aleurone cell walls was not uniform, which is consistent with the studies presented below. Saadi et al. (1998) showed that autofluorescence was more intense in the anticline than in the periclinal cell walls of the corn grains [45]. Moreover, studies have shown that the content of ferulic acid in the anticlinal cell wall of the corn was twice as high as in the periclinal cell wall [46]. However, research by Phillippe et al. [34] argues that anticlinal and periclinal cell walls contain equal amounts of feruloylated arabinoxylan. Therefore, it seems that autofluorescence in the walls of anticlinal aleurone cells can additionally be caused by other substances, for example, coumaric and diferulic acids, which were found in aleurone cells [37].
Our study showed the metabolic profile of the corn Zea mays L. (var. Pioneer) represented as 56 compounds including 2 compounds identified in corn grains for the first time-namely, oxylipins 13-trihydroxy-octadecenoic acid and 9,12,13-trihydroxy-trans-10octadecenoic acid. Laser microscopy showed the presence of polyphenolic compounds and, in particular, hydroxycinnamic and ferulic acids, and anthocyanins, in the tissues of corn grain.
The method used in this study is effective for rapid analysis of the distribution of polyphenolic compounds in seeds and grains of different plants. This approach allows the study of plant morphology and the characterization of relevant bioactive phytochemicals using an inexpensive and fast methodology. The characterization of novel corn hybrid genotypes harvested from different geographical areas is a strategic problem and addressing this problem would allow sustainable development of local agriculture.

Materials and Chemicals
As an object of research, we used corn grains Zea mays L., variety Pioneer P1467. The sample was harvested in 2020 in urban-type settlement Kirovsky (Primorsky Krai, Russian Far East) and obtained from a local farmer.
HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from SIEMENS ULTRA clear (SIEMENS Water Technologies, Munich, Germany), and all other chemicals were analytical grade.

Fractional Maceration
Fractional maceration technique was applied to obtain highly concentrated extracts [47]. From 500 g of the sample, 4 g of corn seeds was randomly selected for maceration. The total amount of the extractant (ethyl alcohol of reagent grade) was divided into 3 parts, and the grains were consistently infused with the first, second, and third parts. The solid-solvent ratio was 1:20. The infusion of each part of the extractant lasted 7 days at room temperature.

Liquid Chromatography
HPLC was performed using Shimadzu LC-20 Prominence HPLC (Shimadzu, Kyoto, Japan) equipped with a UV sensor and C18 silica reverse phase column (4.6 × 150 mm, particle size: 2.7 µm) to perform the separation of multicomponent mixtures. The gradient elution program with two mobile phases (A, deionized water; B, acetonitrile with formic acid 0.1% v/v) was as follows: 0-2 min, 0% B; 2-50 min, 0-100% B; control washing 50-60 min 100% B. The entire HPLC analysis was performed with a UV-vis detector SPD-20A (Shimadzu, Kyoto, Japan) at a wavelength of 230 nm for identification of catechin, epicatechin, quercetin, and other compounds [48]; the temperature was 50 • C, and the total flow rate 0.25 mL min −1 . The injection volume was 10 µL. Additionally, liquid chromatography was combined with a mass spectrometric ion trap to identify compounds.

Optical Microscopy
Before the microscopic examination, a longitudinal and transverse dissection of corn grains was performed with MS-2 sled microtome (Tochmedpribor, Ukraine). The obtained sliced corn grains were placed on microscopic cover glass through immersion oil to reduce light refraction by air gaps.
The autofluorescence parameters of a slice of corn grain were determined using an inverted confocal microscope (confocal laser scanning microscopy-CLSM, LSM 800, Carl Zeiss Microscopy GmbH, Berlin, Germany). The autofluorescence spectrum was chosen using lambda scan mode of the confocal microscope, which allows to determine the emission maximum in a specific sample and obtain spectral acquisition. The specimen was excited by each laser separately and two main peaks of autofluorescence were revealed: excitation by a UV laser, 405 nm (solid state, diode, 5mW) with the emission maxima in the ranges 400-470 nm (blue); excitation by a blue laser, 488 nm (solid state, diode, 10 mW) with the emission maximum in 620-700 nm (red). The used power and detector gain for blue and red channels were 5% and 750 V, and 7% and 850 V, respectively.
The images were obtained using objectives Plan-Apochromat 20×/0.8 M27 and Plan-Apochromat 63×/1.40 Oil DIC M27 with 20× and 63× magnification, correspondingly. The zoom factor was 0.5. Airyscan at the SR mode was used to increase resolution. The software ZEN 2.1 (Carl Zeiss Microscopy GmbH, Berlin, Germany) was used for image acquisition.

Conclusions
We determined the qualitative characteristics of secondary metabolites in the tissues of corn Zea mays L. (var. Pioneer). In total, 56 compounds were identified, including 2 compounds identified in corn grains for the first time-namely, oxylipins 13-trihydroxyoctadecenoic acid and 9,12,13-trihydroxy-trans-10-octadecenoic acid.
The combination of these data with fluorescence microscopy data revealed the most probable localization of phenolic and polyphenolic compounds. In addition, confocal microscopy allowed us to assess the localization of hydroxycinnamic and ferulic acids in aleurone cells and embryos and anthocyanin content in pericarp and aleurone cells. The combination of these methods is important for breeding since it allows us to assess whether the genes involved in the synthesis of these substances are expressed only in certain tissues (the aleurone layer, the germ layer, the vitreous endosperm) or in all grain glutes uniformly. In addition, this approach makes it possible to estimate the number and size of storing organelles (granules, chloroplasts, vesicles), since selection is important both in the area of increasing their number and increasing their size. Thus, the combination of these methods allows us to obtain more complete information about the variables under study. In addition, it shows that confocal microscopy can be used to obtain preliminary information during volumetric screenings of varietal samples, which will allow selecting target groups for more detailed analysis much faster and without the use of expensive reagents.

Data Availability Statement:
The data presented in the current study are available in the article.