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Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose

Transgenic Research volume 22, pages 1133–1142 (2013)Cite this article

Abstract

Maize (Zea mays spp. mays) is a staple crop for more than 900 million people. The seeds or kernels provide a rich source of calories because ~70 % of the weight is carbohydrate, mostly in the form of starch. The content and composition of starch are complex traits controlled by many genes, offering multiple potential targets for intervention. We used a multigene engineering approach combining the overexpression of Bt2, Sh2, Sh1 and GbssIIa (to enhance the activity of sucrose synthase, AGPase and granule-bound starch synthase) with the suppression of SbeI and SbeIIb by RNA interference (to reduce the activity of starch branching enzyme). Maize plants expressing all six genes plus the selectable marker showed a 2.8–7.7 % increase in the endosperm starch content and a 37.8–43.7 % increase in the proportion of amylose, which was significant compared to untransformed control plants. We also observed improvements in other agronomic traits, such as a 20.1–34.7 % increase in 100-grain weight, a 13.9–19.0 % increase in ear weight, and larger kernels with a better appearance, presumably reflecting the modified starch structure within the kernels. Our results confirm that multigene engineering applied to the starch biosynthesis pathway can not only modulate the quality and quantity of starch but can also improve starch-dependent agronomic traits.

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References

  • Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta S, Dix PJ, Fauquet C, Huang N, Kohli A, Mooibroek H, Nicholson L, Nguyen TT, Nugent G, Raemakers K, Romano A, Somers DA, Stoger E, Taylor N, Visser R (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breed 15:305–327

    Article  Google Scholar 

  • Ball SG, Morell MK (2003) From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Ann Rev Plant Biol 54:207–233

    Article  CAS  Google Scholar 

  • Ballicora MA, Laughlin MJ, Fu Y, Okita TW, Barry GF, Preiss J (1995) Adenosine 5′-diphosphate-glucose pyrophosphorylase from potato tuber: significance of the N terminus of the small subunit for catalytic properties and heat stability. Plant Physiol 109:245–251

    PubMed  Article  CAS  Google Scholar 

  • Beatty MK, Rahman A, Cao H, Woodman W, Lee M, Myers AM, James MG (1999) Purification and molecular genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from maize. Plant Physiol 119:255–266

    PubMed  Article  CAS  Google Scholar 

  • Blauth SL, Yao Y, Klucinec JD, Shannon JC, Thompson DB, Guilitinan MJ (2001) Identification of mutator insertional mutants of starch-branching enzyme 2a in corn. Plant Physiol 125:1396–1405

    PubMed  Article  CAS  Google Scholar 

  • Blauth SL, Kim KN, Klucinec J, Shannon JC, Thompson D, Guiltinan M (2002) Identification of mutator insertional mutants of starch-branching enzyme 1 (sbe1) in Zea mays L. Plant Mol Biol 48:287–297

    PubMed  Article  CAS  Google Scholar 

  • Boyer CD, Preiss J (1981) Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases. Plant Physiol 67:1141–1145

    PubMed  Article  CAS  Google Scholar 

  • Boyer C, Daniels RR, Shannon JC (1977) Starch granule (amyloplast) development in endosperm of several Zea mays L. genotypes affecting kernel polysaccharides. Am J Bot 64:50–56

    Article  Google Scholar 

  • 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 12:223

    PubMed  Article  CAS  Google Scholar 

  • Carlson SJ, Chourey PS (1996) Evidence for plasma membrane-associated forms of sucrose synthase in maize. Mol Gen Genet 252:303–310

    PubMed  Article  CAS  Google Scholar 

  • Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218

    PubMed  Article  CAS  Google Scholar 

  • Dian W, Jiang H, Chen Q, Liu F, Wu P (2003) Cloning and characterization of the granule-bound starch synthase II gene in rice: gene expression is regulated by the nitrogen level, sugar and circadian rhythm. Planta 218:261–268

    PubMed  Article  CAS  Google Scholar 

  • Dong ZY, Wang YM, Zhang ZJ, Shen Y, Lin XY, Ou XF, Han FP, Liu B (2006) Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb. Theor Appl Genet 113:196–205

    Google Scholar 

  • Frame BR, Zhang H, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE, Zhen S, Schnable PS, Wang K (2000) Production of transgenic maize from bombarded Type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol Plant 36:21–29

    Article  Google Scholar 

  • Giroux MJ, Shaw J, Barry G, Cobb BG, Greene T, Okita T, Hannah LC (1996) A single gene mutation that increases maize seed weight. Proc Natl Acad Sci USA 93:5824–5829

    PubMed  Article  CAS  Google Scholar 

  • Guan S, Wang P, Liu H, Liu G, Ma Y, Zhao L (2011) Production of high-amylose maize lines using RNA interference in sbe2a. Afr J Biotechnol 10:15229–15237

    Article  CAS  Google Scholar 

  • Guo SJ, Li JR, Qiao WH, Zhang XS (2006) Analysis of amylose accumulation during seed development in maize. Acta Genet Sin 33:1014–1019

    PubMed  Article  CAS  Google Scholar 

  • Hannah LC, Shaw JR, Giroux MJ, Reyss A, Prioul JL, Bae JM, Lee JY (2001) Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase. Plant Physiol 127:173–183

    PubMed  Article  CAS  Google Scholar 

  • Hannah LC, Futch B, Bing J, Shaw JR, Boehlein S, Stewart JD, Beiriger R, Georgelis N, Greene T (2012) A shrunken-2 transgene increases maize yield by acting in maternal tissues to increase the frequency of seed development. Plant Cell 24:2352–2363

    PubMed  Article  CAS  Google Scholar 

  • Hardin SC, Duncan KA, Huber SC (2006) Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1. Plant Physiol 141:1106–1119

    PubMed  Article  CAS  Google Scholar 

  • Hirose T, Terao T (2004) A comprehensive expression analysis of the starch synthase gene family in rice (Oryza sativa L.). Planta 220:9–16

    PubMed  Article  CAS  Google Scholar 

  • Hovenkamp-Hermelink JHM, De Vries JN, Adamse P, Jacobsen E, Witholt B, Feenstra WJ (1988) Rapid estimation of the amylose/amylopectin ratio in small amounts of tuber and leaf tissue of the potato. Potato Res 31:241–246

    Article  CAS  Google Scholar 

  • Jeon JS, Ryoo N, Hahn TR, Walia H, Nakamura Y (2010) Starch biosynthesis in cereal endosperm. Plant Physiol Biochem 48:383–392

    PubMed  Article  CAS  Google Scholar 

  • Jobling SA, Schwall GP, Westcott RJ, Sidebottom CM, Debet M, Gidley MJ, Jeffcoat R, Safford R (1999) A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A. Plant J 18:163–171

    PubMed  Article  CAS  Google Scholar 

  • Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52:247–258

    PubMed  Article  CAS  Google Scholar 

  • Kohli A, Melendi PG, Abranches R, Capell T, Stoger E, Christou P (2006) The quest to understand the basis and mechanisms that control expression of introduced transgenes in crop plants. Plant Signal Behav 1:185–195

    PubMed  Article  Google Scholar 

  • Lee SM, Ryu TH, Kim SI, Okita T, Kim D (2009) Kinetic and regulatory properties of plant ADP-glucose pyrophosphorylase genetically modified by heterologous expression of potato upreg mutants in vitro and in vivo. Plant Cell, Tissue Organ Cult 96:161–170

    Article  CAS  Google Scholar 

  • 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 233:241–250

    PubMed  Article  CAS  Google Scholar 

  • Liu F, Makhmoudova A, Lee EA, Wait R, Emes MJ, Tetlow IJ (2009) The amylose extender mutant of maize conditions novel protein–protein interactions between starch biosynthetic enzymes in amyloplasts. J Exp Bot 60:4423–4440

    PubMed  Article  CAS  Google Scholar 

  • Mehlo L, Mazithulela G, Twyman RM, Boulton MI, Davies JW, Christou P (2000) Structural analysis of transgene rearrangements and effects on expression in transgenic maize plants generated by particle bombardment. Maydica 45:277–287

    Google Scholar 

  • Naqvi S, Farré G, Sanahuja G, Capell T, Zhu C, Christou P (2010) When more is better: multigene engineering in plants. Trends Plant Sci 15:48–56

    PubMed  Article  CAS  Google Scholar 

  • Obana Y, Omoto D, Kato C, Matsumoto K, Nagai Y, Kavakli IH, Hamada S, Edwards GE, Okita TW, Matsui H, Ito H (2006) Enhanced turnover of transitory starch by expression of up-regulated ADP-glucose pyrophosphorylases in Arabidopsis thaliana. Plant Sci 170:1–11

    Article  CAS  Google Scholar 

  • Regina A, Kosar-Hashemi B, Ling S, Li Z, Rahman S, Morell M (2010) Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb. J Exp Bot 61:1469–1482

    PubMed  Article  CAS  Google Scholar 

  • Röper H (2002) Renewable raw materials in Europe—industrial utilisation of starch and sugar. Starch/Staerke 54:89

    Article  Google Scholar 

  • Russell DA, DeBoer DL, Stark DM, Preiss J, Fromm ME (1993) Plastid targeting of β-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells. Plant Cell Rep 13:287–292

    Article  Google Scholar 

  • Safford R, Jobling SA, Sidebottom CM, Westcott RJ, Cooke D, Tober KJ, Strongitharm BH, Russell AL, Gidley MJ (1998) Consequences of antisense RNA inhibition of starch branching enzyme activity on properties of potato starch. Carbohydr Polym 35:155–168

    Article  CAS  Google Scholar 

  • Sano Y (1984) Differential regulation of waxy gene expression in rice endosperm. Theor Appl Genet 68:467–473

    Article  CAS  Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108

    PubMed  Article  CAS  Google Scholar 

  • Schwall GP, Safford R, Westcott RJ, Jeffcoat R, Tayal A, Shi YC, Gidley MJ, Jobling SA (2000) Production of very-high-amylose potato starch by inhibition of SBE A and B. Nat Biotechnol 18:551–554

    PubMed  Article  CAS  Google Scholar 

  • 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 10:144

    PubMed  Article  Google Scholar 

  • Slattery CJ, Kavakli IH, Okita TW (2000) Engineering starch for increased quantity and quality. Trends Plant Sci 5:291–298

    PubMed  Article  CAS  Google Scholar 

  • 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 99:1724–1729

    PubMed  Article  CAS  Google Scholar 

  • Smidansky ED, Martin JM, Hannah LC, Fischer AM, Giroux MJ (2003) Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase. Planta 216:656–664

    PubMed  CAS  Google Scholar 

  • Smith AM (2008) Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54:546–558

    PubMed  Article  CAS  Google Scholar 

  • Stark DM, Timmerman KP, Barry GF, Preiss J, Kishore GM (1992) Regulation of the amount of starch in plant tissues by ADP glucose pyrophosphorylase. Science 258:287–292

    PubMed  Article  CAS  Google Scholar 

  • Tsai CY (1974) The function of the Waxy locus in starch synthesis in maize endosperm. Biochem Genet 11:83–96

    PubMed  Article  CAS  Google Scholar 

  • Vrinten PL, Nakamura T (2000) Wheat granule-bound starch synthase I and II are encoded by separate genes that are expressed in different tissues. Plant Physiol 122:255–263

    PubMed  Article  CAS  Google Scholar 

  • Wei C, Qin F, Zhu L, Zhou W, Chen Y, Wang Y, Gu M, Liu Q (2010a) Microstructure and ultrastructure of high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. J Agric Food Chem 58:1224–1232

    PubMed  Article  CAS  Google Scholar 

  • Wei C, Xu B, Qin F, Yu H, Chen C, Meng X, Zhu L, Wang Y, Gu M, Liu Q (2010b) C-type starch from high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. J Agric Food Chem 58:7383–7388

    PubMed  Article  CAS  Google Scholar 

  • Wu L, Nandi S, Chen L, Rodriguez RL, Huang N (2002) Expression and inheritance of nine transgenes in rice. Transgenic Res 11:533–541

    PubMed  Article  CAS  Google Scholar 

  • Zeeman SC, Kossmann J, Smith AM (2010) Starch: its metabolism, evolution, and biotechnological modification in plants. Annu Rev Plant Biol 61:209–234

    PubMed  Article  CAS  Google Scholar 

  • Zhu C, Naqvi S, Breitenbach J, Sandmann G, Christou P, Capell T (2008) Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proc Natl Acad Sci USA 105:18232–18237

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Transgenic Maize Project #2009ZX08003-024B and Nature Science Foundation of Jilin Province #20080567 (to JP), and the National Nature Science Foundation #31170259 (to XQ), and #31070269 (to CZ). We thank Dr. Robert Henry from Southern Cross University Australia, for kindly providing the endosperm-specific promoters, and to Prof. Paul Christou of Universitat de Lleida for his constructive comments which improved the manuscript.

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Affiliations

  1. Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, 130024, China

    Lili Jiang, Xiaoming Yu, Qian Yu, Sen Deng, Bing Bai, Ning Li, Ai Zhang, Bao Liu & Jinsong Pang

  2. Faculty of Agronomy, Jilin Agricultural University, Changchun, 130118, China

    Xin Qi

  3. School of Life Sciences, Changchun Normal University, Changchun, 130032, China

    Changfu Zhu

  4. Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida-Agrotecnio Center, Av. Alcalde Rovira Roure, 191, 25198, Lleida, Spain

    Changfu Zhu

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  1. Lili Jiang
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  2. Xiaoming Yu
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  3. Xin Qi
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  4. Qian Yu
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  7. Ning Li
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  8. Ai Zhang
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  9. Changfu Zhu
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  10. Bao Liu
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Correspondence to Changfu Zhu, Bao Liu or Jinsong Pang.

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Lili Jiang and Xiaoming Yu contributed equally to this work.

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11248_2013_9717_MOESM1_ESM.tiff

Fig. S1. Expression and silencing cassettes used for maize transformation. The expression of Sh1, Bt2, Sh2 and GbssIIa was driven by different monocot endosperm-specific promoters: barley B1-hordein, rice glutelin-1, barley ISA and wheat high molecular weight glutenin, respectively. The two RNAi constructs were driven by a barley D-hordein promoter. The bar gene was controlled by the maize ubiquitin promoter (Ubi). (TIFF 1148 kb)

11248_2013_9717_MOESM2_ESM.tif

Fig. S2. PCR analysis of genomic DNA from T1 plants transformed with expression cassettes containing Sh1 (a), Bt2 (b), Sh2 (c), GbssIIa (d), SbeI (e) and SbeIIb (f). (TIFF 1575 kb)

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Jiang, L., Yu, X., Qi, X. et al. Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose. Transgenic Res 22, 1133–1142 (2013). https://doi.org/10.1007/s11248-013-9717-4

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  • DOI: https://doi.org/10.1007/s11248-013-9717-4

Keywords

  • Maize (Zea mays)
  • Starch
  • Amylose
  • Genetic engineering
  • Multigene transformation
  • Transgenic plants