A histochemical assay for polyphenolic profiling in cereal grains

In different cell layers, cereal grains may accumulate various economically important polyphenols such as colored anthocyanins and melanins and colorless proanthocyanidins. To effectively create new cultivars with different combinations of these compounds, a simple, fast, and precise screening method is required. Here, a histochemical assay that includes a combination of hot ethanolic, acidic, alkaline, and ammoniacal silver treatments of grain cryosections followed by microscopy was successfully applied to distinguish these substances in cereal grains. Barley lines previously characterized chemically for the presence of anthocyanins, proanthocyanidins, and melanins in grains were used as a model. In black barley grains, this approach allowed to visually distinguish insoluble melanins that do not react to a pH change from anthocyanins, which can be insoluble or soluble but always react to changing pH. For the first time, ammoniacal silver staining commonly used for melanin identification in human and animal tissues was adapted for melanin identification in plant tissues. Along with melanins, this reagent stains other polyphenols thereby helping to detect colorless polyphenols including proanthocyanidins in the testa of barley grains as confirmed by p-dimethylaminocinnamaldehyde (DMACA) staining. The applicability of this assay to polyphenol profiling was demonstrated not only in the barley grain but also in wheat and common vetch grains. The proposed histochemical assay allows rapid polyphenol screening using a single grain, making it a practical and efficient alternative to time-consuming chromatographic methods for preliminary selection from large sample sets prior to detailed quantitative and qualitative chemical analysis.

1. Aastrup S., Outtrup H., Erdal K. Location of the proanthocyanidins in the barley grain. Carlsberg Res Commun. 1984;49:105-109. doi 10.1007/BF02913969

2. Abdel-Aal el-S.M., Young J.C., Rabalski I. Anthocyanin composition in black, blue, pink, purple, and red cereal grains. J Agric Food Chem. 2006;54(13):4696-4704. doi 10.1021/jf0606609

3. Abdel-Aal el-S.M., Hucl P., Shipp J., Rabalski I. Compositional differences in anthocyanins from blue- and purple-grained spring wheat grown in four environments in central Saskatchewan. Cereal Chem J. 2016;93(1):32-38. doi 10.1094/CCHEM-03-15-0058-R

4. Abeynayake S.W., Panter S., Mouradov A., Spangenberg G. A highresolution method for the localization of proanthocyanidins in plant tissues. Plant Methods. 2011;7(1):13. doi 10.1186/1746-4811-7-13

5. Bancroft J.D., Gamble M. (Eds) Theory and Practice of Histological Techniques. Edinburgh: Churchill Livingstone, 2008. doi 10.1016/B978-0-443-10279-0.50001-4

6. Barron C., Holopainen-Mantila U., Sahlstrom S., Hotekjolen A.K., Lullien-Pellerin V. Assessment of biochemical markers identified in wheat for monitoring barley grain tissue. J Cereal Sci. 2017;74: 11-18. doi 10.1016/j.jcs.2017.01.004

7. Bell A.A., Wheeler M.H. Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol. 1986;24:411-451. doi 10.1146/annurev.py.24.090186.002211

8. Britton G. The Biochemistry of Natural Pigments. Cambridge Univ. Press, 1983 Davies K.M. Modifying anthocyanin production in flowers. In: Winefield C., Davies K., Gould K. (Eds) Anthocyanins: Biosynthesis, Functions, and Applications. Springer, 2009;49-80. doi 10.1007/978-0-387-77335-3_3

9. Debeaujon I., Léon-Kloosterziel K.M., Koornneef M. Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant Physiol. 2000;122(2):403-414. doi 10.1104/pp.122.2.403

10. Druka A., Franckowiak J., Lundqvist U., Bonar N., Alexander J., Houston K., Radovic S., Shahinnia F., Vendramin V., Morgante M., Stein N., Waugh R. Genetic dissection of barley morphology and development. Plant Physiol. 2011;155(2):617-627. doi 10.1104/pp.110.166249

11. ElMasry G., Mandour N., Al-Rejaie S., Belin E., Rousseau D. Recent applications of multispectral imaging in seed phenotyping and quality monitoring – an overview. Sensors. 2019;19(5):1090. doi 10.3390/s19051090

12. Ficco D.B.M., De Simone V., Colecchia S.A., Pecorella I., Platani C., Nigro F., Finocchiaro F., Papa R., De Vita P. Genetic variability in anthocyanin composition and nutritional properties of blue, purple, and red bread (Triticum aestivum L.) and durum (Triticum turgidum L. ssp. turgidum convar. durum) wheats. J Agric Food Chem. 2014;62(34):8686-8695. doi 10.1021/jf5003683

13. Gardner R.O. Vanillin-hydrochloric acid as a histochemical test for tannin. Stain Technol. 1975;50(5):315-317. doi 10.3109/10520297509117081

14. Glagoleva A.Y., Shmakov N.A., Shoeva O.Y., Vasiliev G.V., Shatskaya N.V., Börner A., Afonnikov D.A., Khlestkina E.K. Metabolic pathways and genes identified by RNA-seq analysis of barley nearisogenic lines differing by allelic state of the Black lemma and pericarp (Blp) gene. BMC Plant Biol. 2017;17:182. doi 10.1186/s12870-017-1124-1

15. Glagoleva A.Y., Shoeva O.Y., Khlestkina E.K. Melanin pigment in plants: current knowledge and future perspectives. Front Plant Sci. 2020;11:770. doi 10.3389/fpls.2020.00770

16. Glagoleva A., Kukoeva T., Mursalimov S., Khlestkina E., Shoeva O. Effects of combining the genes controlling anthocyanin and melanin synthesis in the barley grain on pigment accumulation and plant development. Agronomy. 2022a;12(1):112. doi 10.3390/agronomy12010112

17. Glagoleva A.Y., Novokreschenov L.A., Shoeva O.Y., Kovaleva O.N., Khlestkina E.K. Studying grain color diversity in the barley collection of VIR. Proc Appl Bot Genet Breed. 2022b;183(3):76-84. doi 10.30901/2227-8834-2022-3-76-84 (in Russian)

18. Glagoleva A.Y., Vikhorev A.V., Shmakov N.A., Morozov S.V., Chernyak E.I., Vasiliev G.V., Shatskaya N.V., Khlestkina E.K., Shoeva O.Y. Features of activity of the phenylpropanoid biosynthesis pathway in melanin-accumulating barley grains. Front Plant Sci. 2022c;13:923717. doi 10.3389/fpls.2022.923717

19. Goncharova A.V. Spring common vetch sowing cultivar Obskaya 16. Pisma v Vavilovskii Zhurnal Genetiki i Selektsii = Lett Vavilov J Genet Breed. 2020;6(1):15-17. doi 10.18699/Letters2020-6-03 (in Russian)

20. Gordeeva E., Shoeva O., Mursalimov S., Adonina I., Khlestkina E. Fine points of marker-assisted pyramiding of anthocyanin biosynthesis regulatory genes for the creation of black-grained bread wheat (Triticum aestivum L.) lines. Agronomy. 2022;12:2934. doi 10.3390/agronomy12122934

21. Jende-Strid B. Genetic control of flavonoid biosynthesis in barley. Hereditas. 1993;119(2):187-204. doi 10.1111/j.1601-5223.1993.00187.x

22. Kiernan J. Histological and Histochemical Methods: Theory and Practice. Cold Spring Harbor, 2008 Knievel D.C., Abdel-Aal el-S.M., Rabalski I., Nakamura T., Hucl P. Grain color development and the inheritance of high anthocyanin blue aleurone and purple pericarp in spring wheat (Triticum aestivum L.). J Cereal Sci. 2009;50(1):113-120. doi 10.1016/j.jcs.2009.03.007

23. Kohyama N., Chono M., Nakagawa H., Matsuo Y., Ono H., Matsunaka H. Flavonoid compounds related to seed coat color of wheat. Biosci Biotechnol Biochem. 2017;81(11):2112-2118. doi 10.1080/09168451.2017.1373589

24. Komyshev E.G., Genaev M.A., Busov I.D., Kozhekin M.V., Artemenko N.V., Glagoleva A.Y., Koval V.S., Afonnikov D.A. Determination of the melanin and anthocyanin content in barley grains by digital image analysis using machine learning methods. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2023;27(7):859-868. doi 10.18699/VJGB-23-99

25. Lee J., Durst R.W., Wrolstad R.E. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method. J AOAC Int. 2005; 88(5):1269-1278. doi 10.1093/jaoac/88.5.1269

26. Li J., Wang B., He Y., Wen L., Nan H., Zheng F., Liu H., Lu S., Wu M., Zhang H. A review of the interaction between anthocyanins and proteins. Food Sci Technol Int. 2021;27(5):470-482. doi 10.1177/1082013220962613

27. Loskutov I.G., Khlestkina E.K. Wheat, barley, and oat breeding for health benefit components in grain. Plants. 2021;10(1):86. doi 10.3390/plants10010086

28. Matseychik I.V., Korpacheva S.M., Lomovsky I.O., Serasutdinova K.R. Prospects for the use of products of processing of buckwheat as functional ingredients. Technol Merchandising Innovative Foodstuff. 2020;61(2):53-57. doi 10.33979/2219-8466-2020-61-2-53-57 (in Russian)

29. Morrison I.N., Kuo J., O’Brien T.P. Histochemistry and fine structure of developing wheat aleurone cells. Planta. 1975;123(2):105-116. doi 10.1007/BF00383859

30. Mursalimov S.R., Goncharova A.V., Glagoleva A.Yu., Shoeva O.Yu. Black seed color of the spring common vetch (Vicia sativa L.) cultivar Obskaya 16 is caused by blue anthocyanins accumulating in macrosclereids. Pisma v Vavilovskii Zhurnal Genetiki i Selektsii = Lett Vavilov J Genet Breed. 2021;7(2):91-95. doi 10.18699/LettersVJ2021-7-10

31. Mursalimov S., Glagoleva A., Khlestkina E., Shoeva O. Chlorophyll deficiency delays but does not prevent melanogenesis in barley seed melanoplasts. Protoplasma. 2022;259(2):317-326. doi 10.1007/s00709-021-01669-3

32. Panato A., Antonini E., Bortolotti F., Ninfali P. The histology of grain caryopses for nutrient location: a comparative study of six cereals. Int J Food Sci Technol. 2017;52(5):1238-1245. doi 10.1111/ijfs.13390

33. Panzella L., Eidenberger T., Napolitano A., d’Ischia M. Black sesame pigment: DPPH assay-guided purification, antioxidant/antinitrosating properties, and identification of a degradative structural marker. J Agric Food Chem. 2012;60(36):8895-8901. doi 10.1021/jf2053096

34. Parker M.L., Ng A., Waldron K.W. The phenolic acid and polysaccharide composition of cell walls of bran layers of mature wheat (Triticum aestivum L. cv. Avalon) grains. J Sci Food Agric. 2005;85(15): 2539-2547. doi 10.1002/jsfa.2304

35. Quideau S., Deffieux D., Douat-Casassus C., Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int. 2011;50(3):586-621. doi 10.1002/anie.201000044

36. Sava V.M., Yang S.-M., Hong M.-Y., Yang P.-C., Huang G.S. Isolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chem. 2001;73(2):177-184. doi 10.1016/S0308-8146(00)00258-2

37. Shoeva O.Y., Mock H.-P., Kukoeva T.V., Börner A., Khlestkina E.K. Regulation of the flavonoid biosynthesis pathway genes in purple and black grains of Hordeum vulgare. PLoS One. 2016;11(10): e0163782. doi 10.1371/journal.pone.0163782

38. Shoeva O.Yu., Strygina K.V., Khlestkina E.K. Genes determining the synthesis of flavonoid and melanin pigments in barley. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2018;22(3): 333-342. doi 10.18699/VJ18.369 (in Russian)

39. Shoeva O.Y., Mursalimov S.R., Gracheva N.V., Glagoleva A.Y., Börner A., Khlestkina E.K. Melanin formation in barley grain occurs within plastids of pericarp and husk cells. Sci Rep. 2020;10:179. doi 10.1038/s41598-019-56982-y

40. Solano F. Melanins: skin pigments and much more – types, structural models, biological functions, and formation routes. New J Sci. 2014; 5:1-28. doi 10.1155/2014/498276

41. Takahashi I., Hara M. Enhancement of starch accumulation in plants by exogenously applied methyl jasmonate. Plant Biotechnol Rep. 2014;8:143-149. doi 10.1007/s11816-013-0304-1

42. Treutter D. Chemical reaction detection of catechins and proanthocyanidins with 4-dimethylaminocinnamaldehyde. J Chromatogr. 1989; 467(1):185-193. doi 10.1016/S0021-9673(01)93963-9

43. Varga M., Berkesi O., Darula Z., May N.V., Palágyi A. Structural characterization of allomelanin from black oat. Phytochemistry. 2016; 130:313-320. doi 10.1016/j.phytochem.2016.07.002

44. Vermerris W., Nicholson R. Phenolic compounds and their effects on human health. In: Phenolic Compound Biochemistry. Springer, 2008;235-255. doi 10.1007/978-1-4020-5164-7_7

45. von Wettstein D. From analysis of mutants to genetic engineering. Annu Rev Plant Biol. 2007;58:1-19. doi 10.1146/annurev.arplant.58.032806.104003

46. Wildi B.S. Silver nitrate as a test for ortho and para dihydric phenols. Science. 1951;113(2929):188. doi 10.1126/science.113.2929.188

47. Winkel-Shirley B. Flavonoids in seeds and grains: physiological function, agronomic importance and the genetics of biosynthesis. Seed Sci Res. 1998;8(4):415-422. doi 10.1017/S0960258500004372

48. Wrolstad R.E., Durst R.W., Lee J. Tracking color and pigment changes in anthocyanin products. Trends Food Sci Technol. 2005;16(9): 423-428. doi 10.1016/j.tifs.2005.03.019

49. Yu R., Wu X., Liu J., Howitt C.A., Bird A.R., Liu C.M., Larkin P.J. Rice with multilayer aleurone: a larger sink for multiple micronutrients. Rice. 2021;14(1):102. doi 10.1186/s12284-021-00543-3

50. Zykin P.A., Andreeva E.A., Tsvetkova N.V., Voylokov A.V. Anatomical and image analysis of grain coloration in rye. Preprints. 2020. doi 10.209