Abstract
Wheat is the major cereal crop in the world. While wheat proteins provide the nutritional requirement for humans, the major concern associated with them is their intolerance among a large genetically predisposed population. These proteins include gluten, albumins like amylase/trypsin inhibitors (ATIs), serpins, thionins, and defensins. In this study, we performed a comparative (bread vs. durum wheat) expression analysis of the genes encoding for different intolerant proteins (IP) during grain development, through transcriptomics approach. Two libraries were generated from each genotype with an average of 103.81 million reads, resulting in 121.3K transcripts for bread wheat; and 75.20 million reads resulting in 117.7K transcripts for durum wheat. RNA-seq results were validated through qRT-PCR. All transcripts related to IPs were extracted; 146 and 133 transcripts were identified in case of bread and durum wheat, respectively. However, only five IP genes were differentially expressed (DEGslog2fold>2) and all of them were classified under the GO terms ‘Molecular Function’. For comparative expression of the IP genes between the two genotypes, fragments per kilobase of exon model per million reads mapped (FPKM) values of each gene were studied. None of the IP transcripts were falling in any pathway, that is because these IPs are synthesized from the genes through transcription followed by translation. These IPs, during grain development, belonged to the major categories of thionin, serpin, ATI, gliadin and glutenin. Bread wheat was expressing a greater number of IP genes and mostly they were upregulated in comparison to the durum wheat. Highest numbers of IP transcripts were mapped on the BB genome; and maximum IP genes were located in chromosome1D. IP genes with higher expression in both the genotypes were further analysed for their stage-specific expression during grain development. Thionins, gliadins and glutenins were mainly expressed at 4–5 weeks after anthesis (WAA); whereas, serpins and ATIs were expressed at 3–4 WAA. The expression of thionin and serpin genes was comparatively low throughout the grain development process, when compared to other IPs. We have also analysed the transcripts related to amylose and amylopectin biosynthesis, where durum wheat showed a high amylose: amylopectin ratio in comparison to bread wheat. Our study through transcriptome analysis is another support for the hypothesis that tetraploid durum wheat is less intolerant and better for consumption for the vulnerable population than that of hexaploid bread wheat.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alaedini A (2021) Molecular triggers of non-celiac wheat sensitivity. Biotechnological strategies for the treatment of gluten intolerance. Elsevier, Amsterdam, pp 25–44. https://doi.org/10.1016/B978-0-12-821594-4.00010-4
Altenbach SB, Chang HC, Yu XB, Seabourn BW, Green PH, Alaedini A (2019) Elimination of omega-1, 2 gliadins from bread wheat (Triticum aestivum) flour: effects on immunogenic potential and end-use quality. Front Plant Sci 10:580. https://doi.org/10.3389/fpls.2019.00580
Altenbach SB, Chang HC, Simon-Buss A (2020) Deciphering the immunogenic potential of wheat flour: a reference map of the salt-soluble proteome from the US wheat Butte 86. Proteome Sci 18(1):1–3. https://doi.org/10.1186/s12953-020-00164-6
Andrews SB (2010) Bioinformatics-FastQC a quality control tool for high throughput sequence data. Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Accessed 6 Dec 2018
Balakireva AV, Zamyatnin AA (2016) Properties of gluten intolerance: gluten structure, evolution, pathogenicity and detoxification capabilities. Nutrients 8(10):644. https://doi.org/10.3390/nu8100644
Banerji A, Ananthanarayan L, Lele SS (2020) The science and technology of chapatti and other indian flatbreads. CRS Press, New York
Baur X, Posch A (1998) Characterized allergens causing bakers’ asthma. Allergy 53(6):562–566. https://doi.org/10.1111/j.1398-9995.1998.tb03931.x
Benbow HR, Jermiin LS, Doohan FM (2019) Serpins: genome-wide characterisation and expression analysis of the serine protease inhibitor family in Triticum aestivum. G3 Genes Genomes Genet 9(8):2709–2722. https://doi.org/10.1534/g3.119.400444
Biesiekierski JR (2017) What is gluten? J Gastroenterol Hepatol 32:78–81. https://doi.org/10.1111/jgh.13703
Bietz JA, Wall JS (1973) Isolation and characterization of gliadin-like subunits from glutenin. Cereal Chem 50:537–547
Brouns F, van Rooy G, Shewry P, Rustgi S, Jonkers D (2019) Adverse reactions to wheat or wheat components. Compr Rev Food Sci Food Saf 18(5):1437–1452. https://doi.org/10.1111/1541-4337.12475
Burkhart A (2016) Pesticide: another possible cause of gluten sensitivity. http://theceliacmd.com/2013/10/pesticides-wheat-gluten-sensitivity-connection/. Accessed 16 June 2021
Call L, Kapeller M, Grausgruber H, Reiter E, Schoenlechner R, D’Amico S (2020) Effects of species and breeding on wheat protein composition. J Cereal Sci 93:102974. https://doi.org/10.1016/j.jcs.2020.102974
Caminero A, Galipeau HJ, McCarville JL, Johnston CW, Bernier SP, Russell AK, Jury J, Herran AR, Casqueiro J, Tye-Din JA, Surette MG (2016) Duodenal bacteria from patients with celiac disease and healthy subjects distinctly affect gluten breakdown and immunogenicity. Gastroenterology 151(4):670–683. https://doi.org/10.1053/j.gastro.2016.06.041
Cebolla Á, Moreno MD, Coto L, Sousa C (2018) Gluten immunogenic peptides as standard for the evaluation of potential harmful prolamin content in food and human specimen. Nutrients 10(12):1927. https://doi.org/10.3390/nu10121927
Di Pede G, Dodi R, Scarpa C, Brighenti F, Dall’Asta M, Scazzina F (2021) Glycemic index values of pasta products: an overview. Foods 10(11):2541. https://doi.org/10.3390/foods10112541
Dipnaik K, Kokare P (2017) Ratio of amylose and amylopectin as indicators of glycaemic index and in vitro enzymatic hydrolysis of starches of long, medium and short grain rice. Int J Res Med Sci 5(10):4502–4505
Garcia DF, Wang Z, Guan J, Yin L, Geng S, Li A, Mao L (2021) Wheatgene: a genomics database for common wheat and its related species. Crop J. https://doi.org/10.1016/j.cj.2021.04.011
Geisslitz S, Shewry P, Brouns F, America AH, Caio GP, Daly M, D’Amico S, De Giorgio R, Gilissen L, Grausgruber H, Huang X (2021) Wheat ATIs: characteristics and role in human disease. Front Nutr 8:265. https://doi.org/10.3389/fnut.2021.667370
Hendrix MM, Foster SL, Cordovado SK (2019) Newborn screening quality assurance program for CFTR mutation detection and gene sequencing to identify cystic fibrosis. J Inborn Errors Metab Screen. https://doi.org/10.1177/2326409816661358
Hogg AC, Gause K, Hofer P, Martin JM, Graybosch RA, Hansen LE, Giroux MJ (2013) Creation of high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa). J Cereal Sci 57(3):377–383. https://doi.org/10.1016/j.jcs.2013.01.001
Holaskova E, Galuszka P, Frebort I, Oz MT (2015) Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology. Biotechnol Adv 33(6):1005–1023. https://doi.org/10.1016/j.biotechadv.2015.03.007
Höng K, Austerlitz T, Bohlmann T, Bohlmann H (2021) The thionin family of antimicrobial peptides. PLoS ONE 16(7):e0254549. https://doi.org/10.1371/journal.pone.0254549
Hüe S, Mention JJ, Monteiro RC, Zhang S, Cellier C, Schmitz J, Verkarre V, Fodil N, Bahram S, Cerf-Bensussan N, Caillat-Zucman S (2004) A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity 21(3):367–377. https://doi.org/10.1016/j.immuni.2004.06.018
Inomata N (2009) Wheat allergy. Curr Opin Allergy Clin Immunol 9(3):238–243. https://doi.org/10.1097/ACI.0b013e32832aa5bc
Junker Y, Zeissig S, Kim SJ, Barisani D, Wieser H, Leffler DA, Zevallos V, Libermann TA, Dillon S, Freitag TL, Kelly CP (2012) Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4. J Exp Med 209(13):2395–2408. https://doi.org/10.1084/jem.20102660
Kaushik M, Rai S, Venkadesan S, Sinha SK, Mohan S, Mandal PK (2020) Transcriptome analysis reveals important candidate genes related to nutrient reservoir, carbohydrate metabolism, and defence proteins during grain development of hexaploid bread wheat and its diploid progenitors. Genes 11(5):509. https://doi.org/10.3390/genes11050509
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12(4):357–360. https://doi.org/10.1038/nmeth.3317
Koning F (2012) Celiac disease: quantity matters. Semin Immunopathol 34(4):541–549. https://doi.org/10.1007/s00281-012-0321-0
Kucek LK, Veenstra LD, Amnuaycheewa P, Sorrells ME (2015) A grounded guide to gluten: how modern genotypes and processing impact wheat sensitivity. Compr Rev Food Sci Food Saf 14(3):285–302. https://doi.org/10.1111/1541-4337.12129
Lasztity R (1984) Wheat proteins. In: Press CPR (ed) The chemistry of cereal proteins. CPR Press, Boca Raton, pp 73–89
Lin J, Gu Y, Bian K (2019) Bulk and surface chemical composition of wheat flour particles of different sizes. J Chem. https://doi.org/10.1155/2019/5101684
Ma F, Li M, Li T, Liu W, Liu Y, Li Y, Hu W, Zheng Q, Wang Y, Li K, Chang J (2013) Overexpression of avenin-like b proteins in bread wheat (Triticum aestivum L.) improves dough mixing properties by their incorporation into glutenin polymers. PLoS ONE 8(7):e66758. https://doi.org/10.1371/journal.pone.0066758
Malik AH (2009) Nutrient uptake, transport and translocation in cereals: influences of environmental and farming conditions. Swed Univ Agric Sci 1:1–46
Mamone G, Picariello G, Addeo F, Ferranti P (2011) Proteomic analysis in allergy and intolerance to wheat products. Expert Rev Proteomics 8(1):95–115. https://doi.org/10.1586/epr.10.98
Mamone G, Nitride C, Picariello G, Addeo F, Ferranti P, Mackie A (2015) Tracking the fate of pasta (T. durum semolina) immunogenic proteins by in vitro simulated digestion. J Agric Food Chem 63(10):2660–2667. https://doi.org/10.1021/jf505461x
Mohan Kumar BV, Prasada Rao UJ, Prabhasankar P (2017) Immunogenicity characterization of hexaploid and tetraploid wheat varieties related to celiac disease and wheat allergy. Food Hydrocoll 28(5):888–903. https://doi.org/10.1080/09540105.2017.1319342
Odintsova TI, Slezina MP, Istomina EA, Korostyleva TV, Kasianov AS, Kovtun AS, Makeev VJ, Shcherbakova LA, Kudryavtsev AM (2019) Defensin-like peptides in wheat analyzed by whole-transcriptome sequencing: a focus on structural diversity and role in induced resistance. PeerJ 7:e6125. https://doi.org/10.7717/peerj.6125
Østergaard H, Rasmussen SK, Roberts TH, Hejgaard J (2000) Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins: cloning and characterization of five major molecular forms. J Biol Chem 275(43):33272–33279. https://doi.org/10.1074/jbc.M004633200
Pastorello EA, Farioli L, Conti A, Pravettoni V, Bonomi S, Iametti S, Fortunato D, Scibilia J, Bindslev-Jensen C, Ballmer-Weber B, Robino AM (2007) Wheat IgE-mediated food allergy in European patients: α-amylase inhibitors, lipid transfer proteins and low-molecular-weight glutenins. Int Arch Allergy Immunol 144(1):10–22. https://doi.org/10.1159/000102609
Picariello G, Mamone G, Cutignano A, Fontana A, Zurlo L, Addeo F, Ferranti P (2015) Proteomics, peptidomics, and immunogenic potential of wheat beer (Weissbier). J Agric Food Chem 63(13):3579–3586. https://doi.org/10.1021/acs.jafc.5b00631
Regina A, Bird A, Topping D, Bowden S, Freeman J, Barsby T, Kosar-Hashemi B, Li Z, Rahman S, Morell M (2006) High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats. Proc Natl Acad Sci USA 103(10):3546–3551. https://doi.org/10.1073/pnas.0510737103
Ribeiro M, Nunes FM (2019) We might have got it wrong: modern wheat is not more toxic for celiac patients. Food Chem 278:820–822. https://doi.org/10.1016/j.foodchem.2018.12.003
Ruiz-Carnicer Á, Comino I, Segura V, Ozuna CV, Moreno MD, López-Casado MÁ, Torres MI, Barro F, Sousa C (2019) Celiac immunogenic potential of α-gliadin epitope variants from Triticum and Aegilops species. Nutrients 11(2):220. https://doi.org/10.3390/nu11020220
Sagu ST, Zimmermann L, Landgräber E, Homann T, Huschek G, Özpinar H, Schweigert FJ, Rawel HM (2020) Comprehensive characterization and relative quantification of α-amylase/trypsin inhibitors from wheat cultivars by targeted HPLC-MS/MS. Foods 9(10):1448. https://doi.org/10.3390/foods9101448
Salentijn EM, Goryunova SV, Bas N, van der Meer IM, van den Broeck HC, Bastien T, Gilissen LJ, Smulders MJ (2009) Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins from homoeologous Gli-2 loci. BMC Genomics 10(1):1–4. https://doi.org/10.1186/1471-2164-10-48
Sander I, Rihs HP, Doekes G, Quirce S, Krop E, Rozynek P, van Kampen V, Merget R, Meurer U, Brüning T, Raulf M (2015) Component-resolved diagnosis of baker’s allergy based on specific IgE to recombinant wheat flour proteins. J Allergy Clin Immunol 135(6):1529–1537. https://doi.org/10.1016/j.jaci.2014.11.021
Schuppan D, Pickert G, Ashfaq-Khan M, Zevallos V (2015) Non-celiac wheat sensitivity: differential diagnosis, triggers and implications. Best Pract Res Clin Gastroenterol 29(3):469–476. https://doi.org/10.1016/j.bpg.2015.04.002
Seifert O, Bayat A, Geffers R, Dienus K, Buer J, Löfgren S, Matussek A (2008) Identification of unique gene expression patterns within different lesional sites of keloids. Wound Repair Regener 16(2):254–265. https://doi.org/10.1111/j.1524-475X.2007.00343.x
Shankar R, Bhattacharjee A, Jain M (2016) Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses. Sci Rep 6(1):1–5. https://doi.org/10.1038/srep23719
Sharma P, Saini M, Sheoran S, Sharma I (2013) Molecular analysis of dimeric α-amylase inhibitor genes in Indian wheat. Indian J Biotechnol 12:153–160
Shewry PR, Halford NG, Lafiandra D (2003) Genetics of wheat gluten proteins. Adv Genet 1(49):111–184. https://doi.org/10.1016/S0065-2660(03)01003-4
Siddiqi RA, Singh TP, Rani M, Sogi DS (2021) Electrophoretic characterization and proportion of different protein fractions in wheat cultivars of North-India. J Agric Food Res 1(4):100137. https://doi.org/10.1016/j.jafr.2021.100137
Singh P, Makharia GK (2020) Food allergy and food hypersensitivity. Clin Basic Neurogastroenterol Motil. https://doi.org/10.1016/B978-0-12-813037-7.00027-3
Stepniak D, Koning F (2006) Celiac disease—sandwiched between innate and adaptive immunity. Hum Immunol 67(6):460–468. https://doi.org/10.1016/j.humimm.2006.03.011
Sultana A, Luo H, Ramakrishna S (2021) Antimicrobial peptides and their applications in biomedical sector. Antibiotics 10(9):1094. https://doi.org/10.3390/antibiotics10091094
Talley NJ (2019) Allergies and irritable bowel syndrome. Gastroenterol Hepatol 15(11):619
Tatham AS, Shewry PR (2008) Allergens to wheat and related cereals. Clin Exp Allergy 38(11):1712. https://doi.org/10.1111/j.1365-2222.2008.03101.x
Tchewonpi Sagu S, Huschek G, Bönick J, Homann T, Rawel HM (2019) A new approach of extraction of α-amylase/trypsin inhibitors from wheat (Triticum aestivum L.), based on optimization using Plackett-Burman and Box-Behnken designs. Molecules 24(19):3589. https://doi.org/10.3390/molecules24193589
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28(5):511–515. https://doi.org/10.1038/nbt.1621
Wrigley CW, Corke H, Seetharaman K, Faubion J (eds) (2015) Encyclopedia of food grains, 2nd edn. Elsevier, Amsterdam
Zevallos VF, Raker V, Tenzer S, Jimenez-Calvente C, Ashfaq-Khan M, Rüssel N, Pickert G, Schild H, Steinbrink K, Schuppan D (2017) Nutritional wheat amylase-trypsin inhibitors promote intestinal inflammation via activation of myeloid cells. Gastroenterology 152(5):1100–1113. https://doi.org/10.1053/j.gastro.2016.12.006
Žilić S, Barać M, Pešić M, Dodig D, Ignjatović-Micić D (2011) Characterization of proteins from grain of different bread and durum wheat genotypes. Int J Mol Sci 12(9):5878–5894. https://doi.org/10.3390/ijms12095878
Acknowledgements
Authors acknowledge the financial support by CRP on Biofortification, Indian Council of Agricultural Research (ICAR) and Department of Biotechnology, Govt. of India for carrying out the work. Authors would also like to acknowledge the support and guidance provided by the Director, ICAR-National Institute for Plant Biotechnology, New Delhi.
Funding
Research work for this study was funded by Indian Council of Agricultural Research—Consortium Research Project on Biofortification vide Sanction Order F.No. CS/F.No.16-8/2017-IA IV dated 26th October 2017 and Department of Biotechnology, Govt. of India vide Sanction Order No. BT/PR7469/FNS/20/733/2013 dated 10th September 2014.
Author information
Authors and Affiliations
Contributions
Conceptualization, PKM and MK; Data curation, PKM; Formal analysis, MK and EM; Funding acquisition, PKM; Investigation, PKM; Methodology, MK; Project administration, PKM; Resources, PKM; Software, MK, EM; Supervision, PKM; Validation, MK; Visualization, PKM GM, AM; Writing—original draft, MK; Writing—review and editing PKM, AM, GM, SM, MK, EM. All authors contributed to the article and approved the submitted version.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no financial and non-financial competing interest.
Additional information
Handling Editor: Weiwei Qi.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Kaushik, M., Mulani, E., Mahendru-Singh, A. et al. Comparative Expression Profile of Genes Encoding Intolerant Proteins in Bread vs. Durum Wheat During Grain Development. J Plant Growth Regul 42, 3200–3210 (2023). https://doi.org/10.1007/s00344-022-10785-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00344-022-10785-0