Metabolomic Proling of Five Hulless Barley with Different Seed Coat Colors

Colored hulless barley (Hordeum vulgare L.) is a high-quality germplasm resource that is rich in nutrients, such as protein, β-glucan, avonoids, amino acids, vitamins, microelements, and dietary ber. However, a systematic evaluation of the metabolites present in colored cultivars is lacking. This study employed an untargeted metabolomics approach using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the metabolic proles of ve hulless barley cultivars with different grain colors. Six-hundred and eight metabolites in various chemical categories were detected; ABC transporters, avonoid biosynthesis, and anthocyanin biosynthesis were found to be the most signicant metabolic pathways. Principal component analysis and orthogonal partial least squares-discriminant analysis revealed signicant metabolic differences among the samples, and the colored barley cultivars could be separated from white barley. Black, blue, and purple grains rich in natural antioxidants were identied as promising ingredients for the development of cereal-based functional foods. These ndings showed that the nutritional function and quality of hulless barley were strongly correlated with its natural color, thereby improving our understanding of the metabolic mechanisms and functional health value associated with different seed coat colors in different hulless barley cultivars. Our results thus provide a theoretical basis for the future study of hulless barley-related products. The results showed that, except for BQ_2 (26/62 in 1127 vs. BQ_2), the avonoids (69/84 in 1127 vs. GZ_160505; in 1127 vs. GZL; and 62/76 in vs. XLQK) were up-regulated as the color of the seed coat changed from white to purple-black. Many avonoids were up-regulated in the colored hulless barley, indicating that colored hulless barley is rich in avonoids.

Therefore, there has been increasing focus on the utilization of colored barley cultivars (Kim et al. 2007; Lin et al. 2018). The most studied seed coat colors are yellow, purple, red, and blue, which are thought to be associated with different subgroups of avonoids (Shoeva et al. 2016).
The seed coat of barley is yellow due to the synthesis of proanthocyanidins in the seed coat (testa layer) (Aastrup et al. 1984); the anthocyanins synthesized in the pericarp and glume are related to red and purple seed coats (Zhang et al. 2019); and anthocyanins synthesized in the aleurone layer of the grain result in a blue seed coat (Harlan. 1914 An untargeted metabolomics approach using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to analyze the metabolic pro les and variations in phytochemicals among these ve different colored hulless barley varieties. Our aim was to contribute toward an elucidation of the metabolic differences among different colored hulless barley cultivars and provide a reference for further nutritional applications of colored hulless barley.

Plant materials
The white cultivar '1127' and blue cultivar 'xiaolanqingke' (XLQK) were collected from Tibet; the blue cultivar 'ganzilan' (GZL) and black-purple cultivar 'ganzi_160505' (GZ-160505) were collected from Ganzi Prefecture in Sichuan Province of China; and the purple cultivar 'beiqing_2' (BQ_2) was collected from Haibei Prefecture in Qinghai Province (Fig. 1A). When the plants were fully mature, they were harvested and dried. Once fully dried, the plants were threshed indoors, and the mature seeds were harvested and stored at − 80°C until further analysis. Grain color was determined by scanning with a color difference meter (Konica Minolta CM-5).

Sample preparation and extraction for metabolomic analysis
The freeze-dried hulless barley seeds were crushed to a powder with a mixer mill (MM 400, Retsch) (30 Hz, 1.5 min). One hundred milligrams of powder were weighed and dissolved in 0.6 mL 70% methanol extract and then dissolved at 4°C overnight. The samples were stored in a bottle for UPLC-MS/MS analysis following centrifugation at 10,000 g for 10 min and then ltered through a 0.22 µm pore membrane.

High-performance liquid chromatography (HPLC) conditions
The data acquisition instrument system included an UPLC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems, 4500 Q TRAP, www.appliedbiosystems.com.cn/). A Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 2.1 mm*100 mm) was used. The mobile phase consisted of solvent A (pure water with 0.04% acetic acid) and solvent B (acetonitrile with 0.04% acetic acid). The gradient program was 95:5 V/V at 0 min, with a linear gradient to 5: 95 V/V over 10 min, where it was held for 1 min at 5:95 V/V at 11.0 min, following which it was adjusted to 95% A 5.0% within 0.10 min, where it was held for 2.9 min. The temperature was 40℃ and the injection volume was 4 µL. The e uent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.

ESI-QTRAP-MS/MS
Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a 4500 QTRAP equipped with an electrospray ionization (ESI) Turbo Ion-Spray interface operating in positive and negative ionization mode and controlled by Analyst 1.6.3 software (AB Sciex).
The ESI conditions mainly included: electrospray ionization temperature, 550℃; spray voltage (IS), 5500 V); and ion source curtain gas (CUR), 30 psi. The collys-activated dissociation (CAD) parameter was set to high. In QQQ, each ion pair was scanned based on the optimized declustering potential (DP) and collision energy (CE).
Qualitative and quantitative analysis of metabolites Qualitative analyses of metabolites were carried out using the self-built database MWDB (Metware Biotechnology Co., Ltd. Wuhan, China) and public database of metabolite information. The isotopic signals, the repeated signals of K + ions, Na + ions, NH4 + ions, and the repeated signals of fragments of other larger molecular weight substances were removed. Metabolites were quanti ed using the MRM model. The mass spectrum peak area of all substances was integrated, and the mass spectrum peak of the same metabolite in different samples was integrated and corrected (Markou & Kassomenos. 2010).

Statistical analysis
The seed coat color differences were evaluated by cluster analysis. Cluster analysis is an analytical method that divides samples into several categories by measuring the similarity degree of the samples, ultimately aggregating them into one class step-by-step (Carlos et al. 2010). The color difference value of the hulless barley cultivars was used as the input into the cluster analysis.
All metabolites were analyzed using R software (www.r-project.org). Principal Component Analysis (PCA), hierarchical clustering analysis (HCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to analyze the metabolic data according to previously described methods (Wang et al. 2016). The data were unit variance-scaled prior to the unsupervised PCA.
The Pearson's correlation coe cient (PCC) between the samples was calculated using the 'cor' function in R, and the HCA results of the metabolites were presented in the form of a heatmap with dendrogram.
Variable importance in the projection (VIP) values extracted from the OPLS-DA results ≥ 1 and absolute Log2FC (fold change) ≥ 1 were used to determine the differentially abundant metabolites between groups. The data were log-transformed (log2) and mean-centered prior to OPLS-DA. A permutation test (200 permutations) was performed to con rm that the OPLS-DA model had not been over tted.
The biological signi cance associated with hulless barley color was further evaluated through the functional analysis of metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) compound database (http://www.kegg.jp/kegg/compound/) to annotate identi ed metabolites, and the annotated metabolites were then mapped to the KEGG pathway database (http://www.kegg.jp/kegg/pathway.html). Pathways with signi cantly regulated metabolites were then fed into MSEA (metabolite sets enrichment analysis), and their signi cance was determined based on the P-values of the hypergeometric test.

Analysis of color differences in hulless barley
The color differences in the seed coat of hulless barley are shown as *L*a*b (L* represents the brightness, a* represents the red-green axis, and b* represents the blue-yellow axis) in Table 1, which re ects the signi cant differences in the color characteristics of the ve hulless barley cultivars (Leónet al, 2006). Analysis of the color value showed that the brightness factor L*, red factor a*, and blue factor b* decreased from white hulless barley to colored hulless barley. In the cluster analysis (Fig. 1B), the white hulless barley could be clearly distinguished from the colored hulless barley varieties.  (Fig. 2) showed there were signi cant differences in the metabolite levels of the ve cultivars, with the colored hulless barley varieties being clearly distinguished from the white cultivar. This nding was generally consistent with the dendrogram based on the color difference values (Fig. 1b). GZL and BQ_2, although cultivated in different regions, grouped into one category according to seed coat color and possessed similar metabolite contents. The white cultivar 1127 could be clearly distinguished from the other cultivars. This nding was demonstrated by clustering analysis of the samples and showed that the metabolites of hulless barley were strongly related to seed coat color.
Differential metabolite analysis using PCA The rst two principal components of the PCA score plot were responsible for 51.31% (28.68% for PC1 and 22.63% for PC2) of the variation in the metabolite pro les. There was a clear separation of these ve cultivars, suggesting that each group had a relatively distinct metabolic pro le. The distinct separation of 1127 (white hulless barley) on the left side of the plot from the other four varieties, which clustered on the right side of the plot, con rmed the distinction of white hulless barley from the colored cultivars. The HCA and PCA results suggested that the differences in the metabolite contents might be responsible for the differences in the seed coat color of hulless barley.
Differential metabolite analysis using OPLS-DA OPLS-DA is a multivariate statistical analysis method for supervised pattern recognition that can maximize the differentiation between groups and identify differential metabolites. In this study, OPLS-DA was carried out to further verify the signi cantly different metabolites between the ve hulless barley cultivars.
High predictability (Q2) and strong goodness-of-t (R2X, R2Y) of the OPLS-DA models were observed for comparisons between 1127 and XLQK (R2X = 0.771, R2Y = 1, Q2 = 0.989; Fig. 4A Using the intersection of each comparison group in the Venn diagram (Fig. 5E), a total of 70 common differential metabolites were observed, and each comparison group possessed unique differential metabolites. The Venn diagram in Fig. 5F shows the overlapping and unique metabolites among the comparison group. There were 28 metabolites that were only present in the colored hulless barley, 19 of which were avonoids and three of which were phenolic acids. These metabolites were primarily avonoids and may be considered as representative differential metabolites for colored hulless barley.
To gain further insight into the differences in avonoids between colored and white hulless barley, differential avonoids among the comparison groups were compared (Supplementary Table 3 Based on the intersection of each comparison group (Supplementary Table 3), 14 common differential avonoid metabolites were up-regulated among the comparison groups 1127 vs. BQ_2, 1127 vs. GZ_160505, 1127 vs. GZL, and 1127 vs. XLQK. Rutin was detected in all the samples, though the differences among the colored hulless barley and white cultivar were nonsigni cant. Except for cyanidin 3,5-O-diglucoside and peonidin 3-O-glucoside in BQ_2, most of the anthocyanins were up-regulated in the colored varieties compared with the control. Iso avones are generally exclusively present in legumes, such as soybeans, and play important roles in plant defense and nodulation (Jung et al. 2000;Kim et al. 2007;Shoeva et al. 2016). In the present study, three iso avones were detected in all hulless barley varieties, of which afzelechin (3,5,7,4'-tetrahydroxy avan) was down-regulated and 2'-hydroxyiso avone was up-regulated, whereas aracarpene 2 was down-regulated in BQ_2 and up-regulated in GZ_160505 and XLQK.
Differential metabolic pathways among the samples The differentially abundant metabolites among the ve hulless barley cultivars were annotated using the KEGG database (http://www.genome.jp/kegg/) in order to obtain the detailed pathway information (Fig. 6A (Fig. 6A-D) also indicated that the differential metabolites of the comparison groups were mainly involved in avonoid biosynthesis, avone and avonol biosynthesis, and anthocyanin biosynthesis.

Conclusions
In the present study, we focused on the metabolic diversity, particularly avonoids, of ve hulless barley varieties and annotated 608 metabolites, including avones, amino acids, and phenolic acids. The metabolic pro les of the colored hulless barley varieties could be effectively discriminated from that of the white cultivar, and the differences were mainly attributed to metabolites associated with ABC transporters, avonoid biosynthesis, and anthocyanin biosynthesis pathways. In addition, 18 avonoids and three phenolic acids were detected and quantitated in the colored hulless barley, but not in the white cultivar. With the exception of BQ_2, more avonoids were up-regulated in the colored hulless barley compared to the white cultivar. These changes may be responsible for the observed variability in hulless barley color, including increased amounts of certain phenolic acids or avonoids. The result was similar to the conclusion of a previous study in colored barley germplasm that found that seed coat color was related to the synthesis of anthocyanins (Kim et al. 2007;Shoeva et al. 2016), though we primarily detected avonoids. In conclusion, colored hulless barley cultivars are rich in avonoids and possess speci c metabolite pro les that differ from white hulless barley, and these avonoids also vary between the different colored cultivars. These ndings improve our understanding of the metabolic mechanisms and functional health value associated with different seed coat colors in different hulless barley cultivars.