Improvement of grain sorghum nutritive properties using modern genetic and biotechnological methods

The paper presents a review of the studies on the use of genetic engineering and genome editing tools for improving nutritional properties of sorghum grain. As a result of experiments performed over the past 5-7 years by several research groups, the created transgenic lines carry genetic constructs for RNA silencing of different kafirin sub-classes (prolamins of sorghum grain). The CRISPR/Cas9 genome editing experiments have yielded mutants with deletions and insertions in the signal sequence of the gene encoding the 22 kDa α-kafirin in sorghum. These lines and mutants were characterized by improved in vitro digestibility of grain proteins, altered ultrastructure of protein bodies and an increased content of lysine. RNA silencing of α-kafirin increased the digestibility of proteins of both raw and cooked flour, while silencing of γ-kafirin led to improved digestibility of proteins of only raw flour. The lines with α-kafirin silencing have kernels with the floury endosperm type that discourages their direct commercial use because of fragility and reduced tolerance to fungal contamination; however, these lines can be used as donors of high digestibility trait when crossed with sorghum lines adapted to local conditions to improve their nutritional value. Kernels of the lines with γ-kafirin silencing may have different endosperm types: floury, vitreous, or a modified type with vitreous endosperm interspersed in the floury endosperm. This fact indicates the possibility of producing agronomically important sorghum lines with high kafirin digestibility and hard endosperm. The increased lysine level in kernels of sorghum lines with the suppressed synthesis of kafirins may be caused by rebalancing of protein synthesis in endosperm of developing kernels due to the synthesis of other proteins, including those with a higher content of essential amino acids. Alongside with improving the digestibility of kafirins, the genetic engineering approach allowed the creation of sorghum lines with a high content of provitamin A in grain and its increased stability during long-term storage. The results of these works show that it is promising to use RNA-interference and genome editing for creating sorghum lines with improved nutritional value of grain.

kafirins, RNA silencing, genome editing, in vitro protein digestibility, endosperm, Sorghum bicolor (L.) Moench

1. Bean S.R., Ioerger B.P., Wilson J.D., Tilley M., Rhodes D., Herald T.J. Structure and chemistry of sorghum grain. In: Rooney W. (ed.). Achieving sustainable cultivation of sorghum. Vol. 2. Sorghum utilization around the world. Cambridge, UK: Burleigh Dodds Science Publishing Limited; 2018. p.3-29. DOI: 10.19103/as.2017.0015.21

2. Belide S., Vanhercke T., Petrie J.R., Singh S.P. Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos. Plant Methods. 2017;13:109. DOI: 10.1186/s13007-017-0260-9

3. Belton P.S., Delgadillo I., Halford N.G., Shewry P.R. Kafirin structure and functionality. Journal of Cereal Science. 2006;44:272-286. DOI: 10.1016/j.jcs.2006.05.004

4. Che P., Zhao Z.-Y., Glassman K. et al. Elevated vitamin E content improves all-trans β-carotene accumulation and stability in biofortified sorghum. Proc. Natl. Acad. Sci. USA. 2016;113:11040-11045. DOI: 10.1073/pnas.1605689113

5. Che P., Anand A., Wu E., Sander J.D., Simon M.K., Zhu W., et al. Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application. Plant Biotechnology J. 2018;16:1388–1395. DOI: 10.1111/pbi.12879

6. Chiquito-Almanza E., Ochoa-Zarzosa A., Lуpez-Meza J.E., PecinaQuintero V., Nuñez-Colín C.A., Anaya-López J.L. A new allele of γ-kafirin gene coding for a protein with high lysine content in Mexican white sorghum germplasm. Journal of the Science of Food and Agriculture. 2016;96(10):3342-3350. DOI: 10.1002/jsfa.7513

7. Cremer J.E., Bean S.R., Tilley M.M., Ioerger B.P., Ohm J.B., Kaufman R.C. et al. Grain Sorghum Proteomics: Integrated Approach toward Characterization of Endosperm Storage Proteins in Kafirin Allelic Variants. Journal of Agricultural and Food Chemistry. 2014;62:9819-9831. DOI: 10.1021/jf5022847

8. De Mesa-Stonestreet N.J., Alavi S., Bean S.R. Sorghum proteins: the concentration, isolation, modification, and food applications of kafirins. Journal of Food Science. 2010;75:90-104. DOI: 10.1111/j.1750-3841.2010.01623.x

9. Da Silva L.S. Transgenic sorghum: Effects of altered kafirin synthesis on kafirin polymerisation, protein quality, protein body structure and endosperm texture [PhD Thesis]. South Africa: University of Pretoria, Department of Food Science, Faculty of Natural and Agricultural Sciences; 2012. 144 p.

10. Da Silva L.S., Jung R., Zhao Z.-Y., Glassman K., Taylor J., Taylor J.R. Effect of suppressing the synthesis of different kafirin sub-classes on grain endosperm texture, protein body structure and protein nutritional quality in improved sorghum lines. Journal of Cereal Science. 2011;54:160-167. DOI: 10.1016/j.jcs.2011.04.009

11. Elkonin L.A., Pakhomova N.V. Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. Plant Cell, Tissue and Organ Culture. 2000;61:115-123. DOI: 10.1023/A:1006472418218

12. Elkonin L.A., Domanina I.V., Italyanskaya Yu.V. Genetic engineering as a tool for modification of seed storage proteins and improvement of nutritional value of cereal grain. Agricultural Biology. 2016a;51(1):17-30. DOI: 10.15389/agrobiology.2016.1.17rus

13. Elkonin L.A., Italianskaya J.V., Domanina I.V., Selivanov N.Y., Rakitin A.L., Ravin N.V. Transgenic sorghum with improved digestibility of storage proteins obtained by Agrobacteriummediated transformation. Russian Journal of Plant Physiology. 2016b;63:678-689. DOI: 10.1134/S1021443716050046

14. Elkonin L.A., Italyanskaya Yu.V. In vitro digestibility of storage endosperm proteins of transgenic sorghum plants carrying genetic construct for silencing of the gamma-kafirin gene. Advances in Current Natural Sciences. 2017;12:96-100. DOI: 10.17513/use.36612

15. Ezeogu L.I., Duodu K.G., Taylor J.R.N. Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours. Journal of Cereal Science. 2005;42:33-44. DOI: 10.1016/j.jcs.2005.02.002

16. Godwin I.D., Williams S.B., Pandit P.S., Laidlaw H.K.C. Multifunctional grains for the future: genetic engineering for enhanced and novel cereal quality. In Vitro Cellular and Developmental Biology - Plant. 2009;45:383-399. DOI: 10.1007/s11627-008-9175-5

17. Grootboom A.W., Mkhonza N.L., Mbambo Z., O’Kennedy M.M., da Silva L.S., Taylor J. et al. Co-suppression of synthesis of major α-kafirin sub-class together with γ-kafirin-1 and γ-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum. Plant Cell Reports. 2014;33:521-537. DOI: 10.1007/s00299-013-1556-5.

18. Hansen J., Sato M., Ruedy R., Lo K., Lea D.W., Medina-Elizade M. Global temperature change. Proc. Natl. Acad. Sci. USA. 2006;103:14288-14293. DOI: 10.1073/pnas.0606291103

19. Henley E.C., Taylor J.R.N., Obukosia S.D. The Importance of Dietary Protein in Human Health: Combating Protein Deficiency in Sub-Saharan Africa through Transgenic Biofortified Sorghum. In: Taylor S.L. (ed.) Advances in Food and Nutrition Research. Burlington, USA: Academic Press, 2010; Vol.60. p.21-52. DOI: 10.1016/S1043-4526(10)60002-2

20. Huang S., Frizzi A., Florida C.A., Kruger D.E., Luethy M.H. High lysine and high tryptophan transgenic maize resulting from the reduction of both 19- and 22-kD α-zeins. Plant Molecular Biology. 2006;61:525-535. DOI: 10.1007/s11103-006-0027-6.

21. Kawakatsu T., Hirose S., Yasuda H., Takaiwa F. Reducing rice seed storage protein accumulation leads to changes in nutrient quality and storage organelle formation. Plant Physiology. 2010;154:1842-1854. DOI: 10.1104/pp.110.164343

22. Kuluev B.R., Gumerova G.R., Mikhaylova E.V., Gerashchenkov G.A., Rozhnova N.A., Vershinina Z.R. et al. Delivery of CRISPR/Cas components into higher plant cells for genome editing. Russian Journal of Plant Physiology. 2019;66(5):694-706. DOI: 10.1134/S102144371905011X

23. Kumar T., Dweikat I., Sato S., Ge Z., Nersesian N., Chen H. et al. Modulation of kernel storage proteins in grain sorghum (Sorghum bicolor (L.) Moench). Plant Biotechnology Journal. 2012;10:533-544. DOI: 10.1111/j.1467-7652.2012.00685.x

24. Laidlaw H.K.C., Mace E.S., Williams S.B., Sakrewski K., Mudge A.M., Prentis P.J. et al. Allelic variation of the beta-, gammaand delta-kafirin genes in diverse Sorghum genotypes. Theoretical and Applied Genetics. 2010;121(7):1227-1237. DOI: 10.1007/s00122-010-1383-9

25. Li A., Jia S., Yobi A., Ge Z., Sato S.J., Zhang C. et al. Editing of an alpha-kafirin gene family increases digestibility and protein quality in sorghum. Plant Physiology. 2018;177(4):1425-1438. DOI: 10.1104/pp.18.00200

26. Lowe K., Wu E., Wang N., Hoerster G., Hastings C., Cho M.-J. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell. 2016;28:1998–2015. DOI: 10.1105/tpc.16.00124

27. Mookkan M., Nelson-Vasilchik K., Hague J., Zhang Z.J., Kausch A.P. Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2. Plant Cell Reports. 2017;36:1477–1491. DOI: 10.1007/s00299-017-2169-1

28. Mehlo L., Mbambo Z., Bado S., Lin J., Moagi S.M., Buthelezi S. et al. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum. Mutation Research 2013;749(1-2):66-72. DOI: 10.1016/j.mrfmmm.2013.05.002

29. Mudge S.R., Campbell B.C., Mustapha N.B., Godwin I.D. Genomic Approaches for Improving Grain Quality of Sorghum. In: Rakshit S., Wang Y.-H. (eds.). The Sorghum Genome. Springer International Publishing AG; 2016. p.189-205. DOI: 10.1007/978-3-319-47789-3_10

30. Nirwan R.S., Kothari S.L. High copper levels improve callus induction and plant regeneration in Sorghum bicolor (L.) Moench. In Vitro Cellular and Developmental Biology – Plant. 2003;39:161–164. DOI: 10.1079/IVP2002385

31. Oria M.P., Hamaker B.R., Shull J.M. Resistance of sorghum α-, β- and γ-kafirins to pepsin digestion. Journal of Agricultural and Food Chemistry. 1995;43(8):2148-2153.

32. Oria M.P., Hamaker B.R., Axtell J.D., Huang C.P. A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies. Proc. Natl. Acad. Sci. USA. 2000;97:5065-5070. DOI: 10.1073/pnas.080076297

33. Reichel M., Li J., Millar A.A. Silencing the silencer: strategies to inhibit microRNA activity. Biotechnology Letters. 2011;33(7): 1285-1292. DOI: 10.1007/s10529-011-0590-z

34. Schmidt M.A., Barbazuk W.B., Sandford M., May G., Song Z., Zhou W. et al. Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome. Plant Physiology. 2011;156(1):330-345. DOI: 10.1104/pp.111.173807

35. Shewry P.R. Improving the protein content and composition of cereal grain. Journal of Cereal Science. 2007;46(3):239-250. DOI: 10.1016/j.jcs.2007.06.006

36. Tesso T., Ejeta G., Chandrashekar A., Huang C.-P., Tandjung A., Lewamy M. et al. A novel modified endosperm texture in a mutant high-protein digestibility/high-lysine grain sorghum (Sorghum bicolor (L.) Moench). Cereal Chemistry. 2006;83:194-201. DOI: 10.1094/CC-83-0194

37. Tuttle J.R., Idris A.M., Brown J.K., Haigler C.H., Robertson D. Geminivirus-Mediated Gene Silencing from Cotton Leaf Crumple Virus Is Enhanced by Low Temperature in Cotton. Plant Physiology. 2008;148(1):41-50. DOI: 10.1104/pp.108.123869

38. Von Born P., Bernardo-Faura M., Rubio-Somoza I. An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana. PLoS ONE. 2018;13(2):e0192984. DOI: 10.1371/journal.pone.0192984

39. Weaver C.A., Hamaker B.R., Axtell J.D. Discovery of grain sorghum germplasm with high uncooked and cooked in vitro protein digestibility. Cereal Chemistry. 1998;75:665-670.

40. Wong J.H., Lau T., Cai N., Singh J., Pedersen J.F., Vensel W.H. et al. Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm. Journal of Cereal Science. 2009;49:73-82. DOI: 10.1016/j.jcs.2008.07.013

41. Wu Y., Messing J. Proteome balancing of the maize seed for higher nutritional value. Front Plant Sci. 2014;5:240. DOI: 10.3389/fpls.2014.00240

42. Wu Y., Yuan L., Guo X., Holding D.R., Messing J. Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum. Nature Communications. 2013;4:2217. DOI: 10.1038/ncomms3217

43. Xing H.L., Dong L., Wang Z.P., Zhang H.Y., Han C.Y., Liu B. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants // BMC Plant Biology. 2014;14:327. DOI: 10.1186/s12870-014-0327-y

44. Xu K., Ren C., Liu Z., Zhang T., Zhang T., Li D. et al. Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophiles. Cellular and Molecular Life Sciences. 2015;72:383-399. DOI: 10.1007/s00018-014-1679-z

45. Zhang G., Hamaker B.R. Low α-amylase starch digestibility of cooked sorghum flours and the effect of protein. Cereal Chemistry 1998;75:710-713. DOI: 10.1094/CCHEM.1998.75.5.710

46. Zhao Z.-Y., Glassman K., Sewalt V., Wang N., Miller M., Chang S. et al. Nutritionally improved transgenic sorghum. In: Vasil I.K. (ed.). Plant Biotechnology 2002 and Beyond. Dordrecht, The Netherlands: Springer; 2003. p.413-416. DOI: 10.1007/978-94-017-2679-5_85