Molecular Breeding

, 35:184 | Cite as

Expression patterns of genes involved in starch biosynthesis during seed development in bread wheat (Triticum aestivum)

  • Anuradha Singh
  • Pankaj Kumar
  • Monica Sharma
  • Rakesh Tuli
  • Harcharan S. Dhaliwal
  • Ashok Chaudhury
  • Dharam Pal
  • Joy Roy
Short Communication


In agricultural crops, seed growth is important for high grain yield. Starch contributes about 50–80 % of the dry weight of seed, and its quality affects both processing and nutrition quality. Despite the wider importance of starch metabolism, the genes involved have not been given much attention or exploited for their use in molecular breeding. Therefore, it is of great interest to analyze the expression of genes involved in starch metabolism for improvement of starch-related traits through molecular breeding. In this study, a quantitative gene expression analysis of 25 starch metabolism genes was conducted in three bread wheat (Triticum aestivum) genotypes differing in yield- and starch-related traits at five seed developmental stages, i.e., 7, 14, 21, 28, and 35 days after anthesis. Their sequences were physically mapped to chromosomes using the wheat genome sequence data through in silico analysis. Their expression data showed dynamic variation during seed development in wheat genotypes. The 25 genes were divided into four groups depending on their expression patterns during seed development. For example, one group was characterized by a high level of expression at early and middle stages as exhibited by different isoforms of starch synthases, starch-branching enzymes, isoamylase, and transcription factors (TaRSR1 and SPA). The enzymes of these genes are key factors in starch biosynthesis. The starch metabolism genes with high expression levels will be sequenced in a wheat germplasm set to develop single nucleotide polymorphism markers for improvement of yield- and starch-related traits through molecular breeding approaches.


Bread wheat Triticum aestivum Gene expression qRT-PCR Starch metabolism genes Transcription factors 



We would like to thank the Executive Director, National Agri-Food Biotechnology Institute (NABI), Mohali, India, for providing funds and facility. Anuradha Singh acknowledges Department of Biotechnology (DBT), Government of India, for providing Junior Research Fellowship (JRF) and Senior Research Fellowship (SRF). We acknowledge IIT, Roorkee, India, and IIWBR (earlier DWR), Karnal, India, for supplying wheat genotypes and Prof. Narpinder Singh, GNDU, Amritsar, India, for determination of starch granule size distribution.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Supplementary material

11032_2015_371_MOESM1_ESM.docx (28 kb)
Supplementary material S1 (MS-word) Protocols of starch extraction and amylose estimation and details of primer pairs (5’-3’), designed for quantitative real-time PCR (qRT-PCR) for gene expression studies. (DOCX 28 kb)
11032_2015_371_MOESM2_ESM.docx (192 kb)
Supplementary material S2 (MS-word) Fig. S1 Amylose content (%) (solid line) and fresh weight (g/10 kernels) (dotted line) at five seed developmental stages (7, 14, 21, 28, and 35 days after anthesis, DAA) and mature seeds of the three wheat genotypes, ‘Amylopectin 1D 0630’ (black circle/white circle), C 306 (black square/white square), and K 65 (black triangle/white triangle); Fig. S2 Starch granule size distribution (percentage) in the seeds of the three wheat genotypes, ‘Amylopectin 1D 0630’ (dotted bar), K 65 (crossed bar), and C 306 (slanted bar) using a Malvern Mastersizer (Malvern Instruments Ltd., UK); Fig. S3 Dendogram of 25 starch metabolic genes and related transporters and transcription factors. It was prepared using pairwise genetic distance matrix (Nei 1972) among the genes and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering method of SAHN module in NTSYSpc v2.21q (Rohlf 2000). (DOCX 192 kb)
11032_2015_371_MOESM3_ESM.xlsx (27 kb)
Supplementary material S3a (MS-xlsx) Normalized threshold cycle (ΔCт) of the 25 starch metabolic genes and related transporters and transcription factors at five seed developmental stages, i.e., 7, 14, 21, 28, and 35 days after anthesis (DAA) of the three wheat genotypes, ‘Amylopectin 1D 0630,’ K 65, and ‘C 306.’ (XLSX 26 kb)
11032_2015_371_MOESM4_ESM.docx (135 kb)
Supplementary material S3b (MS-word) Relative expression data (log2 of fold change) of the 21 starch metabolic genes of four groups (I, II, III, and IV) at five seed developmental stages of the three wheat genotypes, ‘Amylopectin 1D 0630’ (dotted bar), K 65 (crossed bar), and C 306 (slanted bar). All data are shown as means ± SD from three technical replicates. ADP-ribosylation factor (ARF) was used for the normalization of gene expression data. The X-axis represents the seed-developing stages in days, i.e., 7, 14, 21, 28, and 35 days after anthesis (DAA), and the Y-axis represents their relative expression (log2 of relative fold change). The ‘*’ on the bars represents the lowest expression value, which was used to prepare relative fold change (log2), and hence, it is zero [log2 (1) = 0]. (DOCX 134 kb)


  1. Abel GJW, Springer F, Willmitzer L, Kossman J (1996) Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.). Plant J 10:981–991CrossRefPubMedGoogle Scholar
  2. Albani D, Hammond-Kosack MCU, Smith C, Conlan S, Colot V, Holdsworth M, Bevan MW (1997) The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes. Plant Cell 9:171–184PubMedCentralCrossRefPubMedGoogle Scholar
  3. Aoki N, Whitfeld P, Hoeren F, Scofield G, Newell K, Patrick J, Offler C, Clarke B, Rahman S, Furbank RT (2002) Three sucrose transporter genes are expressed in the developing grain of hexaploid wheat. Plant Mol Biol 50:453–462CrossRefPubMedGoogle Scholar
  4. Asai H, Abe N, Matsushima R, Crofts N, Oitome NF, Nakamura Y, Fujita N (2014) Deficiencies in both starch synthase IIIa and branching enzyme IIb lead to a significant increase in amylose in SSIIa-inactive japonica rice seeds. J Exp Bot 65:5497–5507PubMedCentralCrossRefPubMedGoogle Scholar
  5. Ball S, Guan H-P, James M, Myers A, Keeling P, Mouille G, Buléon A, Colonna P, Preiss J (1996) From glycogen to amylopectin: a model for the biogenesis of the plant starch granule. Cell 86:349–352CrossRefPubMedGoogle Scholar
  6. Carciofi M, Blennow A, Jensen SL, Shaik SS, Henriksen A, Buléon A, Holm PB, Hebelstrup KH (2012) Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules. BMC Plant Biol 12:223PubMedCentralCrossRefPubMedGoogle Scholar
  7. Fu FF, Xue HW (2010) Coexpression analysis identifies Rice Starch Regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator. Plant Physiol 154:927–938PubMedCentralCrossRefPubMedGoogle Scholar
  8. Fujita N, Kubo A, Suh D-S, Wong K-S, Jane J-L, Ozawa K, Takaiwa F, Inaba Y, Nakamura Y (2003) Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm. Plant Cell Physiol 44:607–618CrossRefPubMedGoogle Scholar
  9. Hazard B, Zhang X, Colasuonno P, Uauy C, Beckles DM, Dubcovsky J (2012) Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat. Crop Sci 52:1754–1766Google Scholar
  10. Hogg AC, Gause K, Hofer P, Martin JM, Graybosch RA, Hansen LE, Giroux MJ (2013) Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa). J Cereal Sci 57:377–383CrossRefGoogle Scholar
  11. Huang B, Hennen-Bierwagen TA, Myers AM (2014) Functions of multiple genes encoding ADP-glucose pyrophosphorylase subunits in maize endosperm, embryo, and leaf. Plant Physiol 164:596–611PubMedCentralCrossRefPubMedGoogle Scholar
  12. Hussain H, Mant A, Seale R, Zeeman S, Hinchliffe E, Edwards A, Hylton C, Bornemann S, Smith AM, Martin C, Bustos R (2003) Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans. Plant Cell 15:133–149PubMedCentralCrossRefPubMedGoogle Scholar
  13. International Wheat Genome Sequencing Consortium [IWGSC] (2014) A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 345:1251788. doi: 10.1126/science.1251788 CrossRefGoogle Scholar
  14. Juliano BO (1971) A simplified assay for milled-rice amylose. Cereal Sci Today 16:334–340Google Scholar
  15. Kang GZ, Xu W, Liu GQ, Peng XQ, Guo TC (2013) Comprehensive analysis of the transcription of starch synthesis genes and the transcription factor RSR1 in wheat (Triticum aestivum) endosperm. Genome 56:115–122CrossRefPubMedGoogle Scholar
  16. Kubo A, Fujita N, Harada K, Matsuda T, Satoh H, Nakamura Y (1999) The starch-debranching enzymes isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm. Plant Physiol 121:399–409PubMedCentralCrossRefPubMedGoogle Scholar
  17. Lafiandra D, Sestili F, D’Ovidio R, Janni M, Botticella E, Ferrazzano G, Silvestri M, Ranieri R, DeAmbrogio E (2010) Approaches for the modification of starch composition in durum wheat. Cereal Chem 87:28–34CrossRefGoogle Scholar
  18. Leterrier M, Holappa LD, Broglie KE, Beckles DM (2008) Cloning, characterization and comparative analysis of a starch synthase IV gene in wheat: functional and evolutionary implications. BMC Plant Biol 8:98PubMedCentralCrossRefPubMedGoogle Scholar
  19. Li N, Zhang S, Zhao Y, Li B, Zhang J (2011) Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize. Planta 233:241–250CrossRefPubMedGoogle Scholar
  20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2[-Delta Delta C(T)] method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  21. McCready RM, Hassid WZ (1943) The separation and quantitative estimation of amylose and amylopectin in potato starch. J Am Chem Soc 65:1154–1157CrossRefGoogle Scholar
  22. McMaugh SJ, Thistleton JL, Anschaw E, Luo J, Konik-Rose C, Wang H, Huang M, Larroque O, Regina A, Jobling SA, Morell MK, Li Z (2014) Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm. J Exp Bot 65:2189–2201PubMedCentralCrossRefPubMedGoogle Scholar
  23. Meyer FD, Talbert LE, Martin JM, Lanning SP, Greene TW, Giroux MJ (2007) Field evaluation of transgenic wheat expressing a modified ADP-glucose pyrophosphorylase large subunit. Crop Sci 47:336–342CrossRefGoogle Scholar
  24. Nei M (1972) Genetic distance between populations. Am Nat 106:283–292CrossRefGoogle Scholar
  25. Ohdan T, Francisco PB Jr, Sawada T, Hirose T, Terao T, Satoh H, Nakamura Y (2005) Expression profiling of genes involved in starch synthesis in sink and source organs of rice. J Exp Bot 56:3229–3244CrossRefPubMedGoogle Scholar
  26. Peng M, Gao M, Abdel-Aal ESM, Hucl P, Chibbar RN (1999) Separation and characterization of A- and B-type starch granules in wheat endosperm. Cereal Chem 76:375–379CrossRefGoogle Scholar
  27. Regina A, Kosar-Hashemi B, Li Z, Pedler A, Mukai Y, Yamamoto M, Gale K, Sharp PJ, Morell MK, Rahman S (2005) Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus. Planta 222:899–909CrossRefPubMedGoogle Scholar
  28. 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 103:3546–3551PubMedCentralCrossRefPubMedGoogle Scholar
  29. Rohlf FJ (2000) NTSYS-pc, numerical taxonomy and multivariate analysis system. Applied Biostatistics Inc., New YorkGoogle Scholar
  30. Roldan I, Wattebled F, Lucas MM, Delvalle D, Planchot V, Jimenez S, Perez R, Ball S, D’Hulst C, Merida A (2007) The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation. Plant J 49:492–504CrossRefPubMedGoogle Scholar
  31. Schupp N, Ziegler P (2004) The relation of starch phosphorylases to starch metabolism in wheat. Plant Cell Physiol 45:1471–1484CrossRefPubMedGoogle Scholar
  32. Sears ER, Miller TE (1985) The history of Chinese spring wheat. Cereal Res Commun 13:261–263Google Scholar
  33. Sestili F, Janni M, Doherty A, Botticella E, D’Ovidio R, Masci S, Jones HD, Lafiandra D (2010) Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biol 10:144PubMedCentralCrossRefPubMedGoogle Scholar
  34. She K-C, Kusano H, Koizumi K, Yamakawa H, Hakata M, Imamura T, Fukuda M, Naito N, Tsurumaki Y, Yaeshima M, Tsuge T, Matsumoto K, Kudoh M, Itoh E, Kikuchi S, Kishimoto N, Yazaki J, Ando T, Yano M, Aoyama T, Sasaki T, Satoh H, Shimada H (2010) A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality. Plant Cell 22:3280–3294PubMedCentralCrossRefPubMedGoogle Scholar
  35. Shewry PR, Underwood C, Wan Y, Lovegrove A, Bhandari D, Toole G, Mills ENC, Denyer K, Mitchell RAC (2009) Storage product synthesis and accumulation in developing grains of wheat. J Cereal Sci 50:106–112CrossRefGoogle Scholar
  36. Singh S, Singh N, Isono N, Noda T (2010) Relationship of granule size distribution and amylopectin structure with pasting, thermal, and retrogradation properties in wheat starch. J Agric Food Chem 58:1180–1188CrossRefPubMedGoogle Scholar
  37. Singh A, Mantri S, Sharma M, Chaudhury A, Tuli R, Roy J (2014) Genome-wide transcriptome study in wheat identified candidate genes related to processing quality, majority of them showing interaction (quality × development) and having temporal and spatial distributions. BMC Genomics 15:29PubMedCentralCrossRefPubMedGoogle Scholar
  38. Slade AJ, McGuire C, Loeffler D, Mullenberg J, Skinner W, Fazio G, Holm A, Brandt KM, SteineMN Goodstal JF, Knauf VC (2012) Development of high amylose wheat through TILLING. BMC Plant Biol 12:69PubMedCentralCrossRefPubMedGoogle Scholar
  39. Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK, Talbert LE, Giroux MJ (2002) Enhanced ADP-glucose pyrophosphorylase activity in wheat endosperm increases seed yield. Proc Natl Acad Sci USA 99:1724–1729PubMedCentralCrossRefPubMedGoogle Scholar
  40. Smith AM, Denyer K, Martin CR (1995) What controls the amount and structure of starch in storage organs?. Plant Physiol 107:673–677PubMedCentralCrossRefPubMedGoogle Scholar
  41. Smith AM, Zeeman SC, Smith SM (2005) Starch degradation. Annu Rev Plant Biol 56:73–98CrossRefPubMedGoogle Scholar
  42. Stamova BS, Laudencia-Chingcuanco D, Beckles DM (2009) Transcriptomic analysis of starch biosynthesis in the developing grain of hexaploid wheat. Int J Plant Genomics 2009:407–426CrossRefGoogle Scholar
  43. Steup M, Robenek H, Melkonian M (1983) In-vitro degradation of starch granules isolated from spinach chloroplasts. Planta 158:428–436CrossRefPubMedGoogle Scholar
  44. Tetlow IJ (2006) Understanding storage starch biosynthesis in plants: a means to quality improvement. Can J Bot 84:1167–1185CrossRefGoogle Scholar
  45. Tickle P, Burrell MM, Coates SA, Emes MJ, Tetlow IJ, Bowsher CG (2009) Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm. J Plant Physiol 166:1465–1478CrossRefPubMedGoogle Scholar
  46. Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, Berardi S, Comai L, Dubcovsky J (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 9:115PubMedCentralCrossRefPubMedGoogle Scholar
  47. Wang JC, Xu H, Zhu Y, Liu QQ, Cai XL (2013) OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm. J Exp Bot 64:3453–3466PubMedCentralCrossRefPubMedGoogle Scholar
  48. Williams PC, Kuzina FD, Hlynka I (1970) A rapid colorimetric procedure for estimating the amylose content of starches and flours. Cereal Chem 47:411–421Google Scholar
  49. Zhu Y, Cai XL, Wang ZY, Hong MM (2003) An interaction between a MYC protein and an EREBP protein is involved in transcriptional regulation of the rice Wx gene. J Biol Chem 278:47803–47811CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Anuradha Singh
    • 1
    • 3
  • Pankaj Kumar
    • 1
  • Monica Sharma
    • 1
  • Rakesh Tuli
    • 1
  • Harcharan S. Dhaliwal
    • 2
  • Ashok Chaudhury
    • 3
  • Dharam Pal
    • 4
  • Joy Roy
    • 1
  1. 1.National Agri-Food Biotechnology Institute (NABI)MohaliIndia
  2. 2.Eternal UniversitySirmourIndia
  3. 3.Department of Bio and Nano Technology, Bio and Nano Technology CentreGuru Jambheshwar University of Science and TechnologyHisarIndia
  4. 4.Regional Station, Tutikandi CentreIndian Agricultural Research Institute (IARI)ShimlaIndia

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