Skip to main content
SpringerLink
Log in

Pyramiding of three C4 specific genes towards yield enhancement in rice

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

C4 plants can efficiently accumulate CO2 in leaves and thus reduce wasteful oxygen fixation by the RuBisCO enzyme. Three C4 enzymes, namely carbonic anhydrase (CA), phosphoenol pyruvate (PEPC) and pyruvate orthophosphate dikinase (PPDK), were over expressed in Oryza sativa L. ssp. indica var. Khitish under the control of green tissue specific promoters PD54o, PEPC and PPDK, respectively. Integration of these genes was confirmed by Southern hybridization. The relative expression of PEPC, CA and PPDK were, respectively, 6.75, 6.57 and 3.6-fold higher in transgenic plants compared to wild type plants (control). Photosynthetic efficiency of the transgenic plants increased significantly along with a 12 % increase in grain yield compared to wild type plants. Compared to control plants, transgenic plants also showed phenotypic changes such as increased leaf blade size, root biomass, and plant height and anatomical changes such as greater leaf vein number, bundle sheath cells, and bulliform cells. Our findings indicate that the combined over expression of these three enzymes is an efficient strategy for incorporating beneficial physiological and anatomical features that will enable subsequent yield enhancement in C3 rice plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

RUE:

Radiation use efficiency

CA:

Carbonic anhydrase

PEPC:

Phosphoenol pyruvate carboxylase

PPDK:

Pyruvate orthophosphate dikinase

MDH:

Malate dehydrogenase

ME:

Maleic enzyme

MCS:

Multiple cloning sites

MS:

Murashige and Skoog

DTT:

Dithiothreitol

PIABS:

Absolute photosynthetic index

References

  • Ali N, Paul S, Gayen D, Sarkar SN, Datta SK, Datta K (2013) Development of low phytate rice by RNAi mediated seed-specific silencing of inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene (IPK1). PLoS One 8(7):e68161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Amane M, Hiroshi S, Jun H, Tadahiko M, Kunihiko O, Barry O (1992) Distinctive responses of ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase in Wheat leaves to nitrogen nutrition and their possible relationships to Co2-Transfer resistance. Plant Physiol 100(4):1737–1743

    Article  Google Scholar 

  • Arnon DL (1949) Copper enzyme is isolated chloroplast. Polyphenol oxidase in Beta Vulgaries. Plant Physiol 24(1):1–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bandyopadhyay A, Datta K, Zhang J, Yang W, Raychaudhuri S, Miyao M, Datta SK (2007) Enhanced photosynthesis rate in genetically engineered indica rice expressing pepc gene cloned from maize. Plant Sci 172(6):1204–1209

    Article  CAS  Google Scholar 

  • Bjorkman B, Demmig (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170(4):489–504

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254

    Article  CAS  PubMed  Google Scholar 

  • Brown RH, Bouton JH (1993) Physiology and genetics of interspecific hybrids between photosynthetic types. Annu Rev Plant Physiol Plant Mol Biol 44:435–456

    Article  Google Scholar 

  • Burgess SJ, Hibberd JM (2015) Insights in to C4 metabolism from comparative deep sequencing. Curr Opin Plant Biol 25:138–144

    Article  CAS  PubMed  Google Scholar 

  • Comar CL (1942) Analysis of plant extracts for chlorophylls a and b using a commercial spectrophotometer. Ind Eng Chem Anal Edit 14 (11): 877–879

    Article  CAS  Google Scholar 

  • Dai Z, Ku MSB, Edwards GE (1993) C4 photosynthesis: the CO2 concentrating mechanism and photorespiration. Plant Physiol 103(1):83–90

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Datta SK, Peterhans A, Datta K, Potrykus I (1990) Genetically engineered fertile indica-rice recovered from protoplasts. Biotechnology 8(8):736–740

    Article  CAS  Google Scholar 

  • Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush G, Datta SK (1998) Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest. Theor Appl Genet 97(1):20–30

    Article  CAS  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1(4):19–21

    Article  CAS  Google Scholar 

  • Ding ZS, Huang SH, Zhou BY (2013) Over-expression of phosphoenolpyruvate carboxylase cDNA from C4 millet (Seteria italica) increase rice photosynthesis and yield under upland condition but not in wetland fields. Plant Biotech Rep 7 (2):155–163

    Article  Google Scholar 

  • Freitag H, Stichler W (2000) A remarkable new leaf type with unusual photosynthetic tissue in a central Asiatic genus of Chenopodiaceae. Plant Biol (2): 154–160

  • Fukayama H, Tsuchida H, Agarie S, Nomura M, Onodera H, Ono K, Lee BH, Hirose S, Toki S, Ku MSB, Makino A, Matsuoka M, Miyao M (2001) Significant accumulation of C4 specific pyruvate, orthophosphate dikinase in a C3 plant rice. Plant Physiol 127(3):1136–1146

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudoh S, Furbank RT, Miyao M (2003) Activity regulation and physiological impacts of the maize C4-specific phosphoenol pyruvate carboxylase overproduced in transgenic rice plants. Photosynth Res 77 (2):227–239

    Article  CAS  PubMed  Google Scholar 

  • Hatch MD (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim Biophys Acta 895:81–106

    Article  CAS  Google Scholar 

  • Hausler RE, Rademacher T, Li J, Lipka V, Fischer KL, Schubert S, Kreuzaler F, Hirsch HJ (2001) Single and double overexpression of C4-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato and tobacco plants. J Exp Bot 52(362):1785–1803

    Article  CAS  PubMed  Google Scholar 

  • Henriques FS (2003) Gas exchange, chlorophyll a fluorescence kinetics and lipid peroxidation of pecan leaves with varying manganese concentrations. Plant Sci 165(1):239–244

    Article  CAS  Google Scholar 

  • Hibberd JM, Sheehy JE, Langdale JA (2008) Using C4 photosynthesis to increase the yield of rice—rationale and feasibility. Curr Opin Plant Biol 11(2):228–231

    Article  CAS  PubMed  Google Scholar 

  • Huang P, Brutnell TP (2016) A synthesis of transcriptomic surveys to dissect the genetic basis of C4 photosynthesis. Curr Opin Plant Biol 31:91–99

    Article  CAS  PubMed  Google Scholar 

  • Huang N, Angeles E, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennett J, Khush G (1997) Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR. Theor Appl Genet 95(3):313–320

    Article  CAS  Google Scholar 

  • HuiFang Z, WeiGang Xu, HuiWei W, Lin Hu, Yan Li, XueLi Qi, Lei Z, ChunXin Li, Xia Hua (2014) Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat. Protoplasma 251(5):1163–1173

    Article  Google Scholar 

  • IRRI (2002) Standard evaluation system for rice (SES). International Rice Research Institute, Manila

    Google Scholar 

  • Jianfu Z, Bandyopadhyay A, Krisnan S, Guoying W, Hua’an X, Datta K, Datta SK (2009) Characterization of a C4 maize pyruvate orthophosphate dikinase expressed in C3 transgenic rice plants. Afr J Biotechnol 9(2):234–242

    Google Scholar 

  • Jiao D, Huang X, Li X, Chi W, Kuang T, Zhang Q, Ku MSB, Cho D (2002) Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes. Photosynth Res 72 (1):85–93

    Article  CAS  PubMed  Google Scholar 

  • Jinheng Z, Chao H, Zhiheng L (2009) Absorption spectrum estimating rice chlorophyll concentration: preliminary investigations. J Plant Breed Crop Sci 1(5):223–229

    Google Scholar 

  • Karmakar S, Molla KA, Chanda PK, Sarkar SN, Datta SK, Datta K (2016) Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight. Planta 243(1):115–130

    Article  CAS  PubMed  Google Scholar 

  • Kate M, Jhonson G (2000) Chlorophyll fluorescence-a practical guide. J Exp Bot 51(345):659–668

    Article  Google Scholar 

  • Kitajima M, Butler WL (1975) Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromthymoquinone. Biochim Biophys Acta 376(1):105–115

    Article  CAS  PubMed  Google Scholar 

  • Ku MSB, Wu J, Dai Z, Scott RA, Chu C, Edwards GE (1991) Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol 96(2):518–528

  • Ku MSB, Sakae A, Mika N, Hiroshi F, Hiroko T, Kazuko O, Sakiko H, Seiichi T, Mitsue M, Makoto M (1999) High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol 17(1): 76–80

    Article  CAS  PubMed  Google Scholar 

  • Ku MSB, Ranade U, Hsu TP, Matsuoka M (2000) Photosynthetic performance of transgenic rice plants overexpressing maize C4 photosynthesis enzymes. Stud. Plant Sci 7:193–204

    Article  CAS  Google Scholar 

  • Ku MSB, Cho D, Ranade U, Hsu TP, Li X, Jiao DM, EhleringerJ, Miyao M, Ludwig M (2012) Carbonic anhydrase and the molecular evolution of C4 photosynthesis. Plant Cell Environ 35(1):22–37

    Article  Google Scholar 

  • Kumar K, Muthamilarasan M, Prasad M (2013) Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions. Plant Cell Tissue Org Cult 115:13–22

    Article  CAS  Google Scholar 

  • Li B, Chen DL, Shi JN (1987) Purification and molecular properties of NADP dependent malate dehydrogenase from sorghum leaves. Acta Photophysiol Sin 13: 113–121

    CAS  Google Scholar 

  • Long SP, Zhu XG, Naidu SL, Ort DR (2006) Can improvement in photosynthesis increase crop yields? Plant Cell Environ 29(3):315–330

    Article  CAS  PubMed  Google Scholar 

  • Ma X, Feng DS, Wang HG, Li XF, Kong LR (2011) Cloning and expression analysis of wheat cytokinin oxidase/dehydrogenase gene TaCKX3. Plant Mol Biol Rep 29(1):98–105

    Article  CAS  Google Scholar 

  • Magnin NC, Cooley BA, Reiskind JB, Bowes G (1997) Regulation and localization of key enzymes during the induction of Kranz-less, C4 photosynthesis in Hydrilla verticillate. Plant Physiol 115(4):1681–1689

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marques da Silva J, Arrabaca MC (2004) Photosynthesis in the water-stressed C4 grass Setaria sphacelata is mainly limited by stomata with both rapidly and slowly imposed water deficits. Physiol Plant 121:409–420

    Article  Google Scholar 

  • Masumoto C, Miyazawa S, Ohkawa H, Fukuda T, Taniguchi Y, Murayama S, Kusano M, Saito K, Fukayama H, Miyao M (2010) Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation. Proc Natl Acad Sci USA 107 (11):5226–5231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Matsuoka M, Furbank RT, HF, Miyao M (2001) Molecular engineering of C4 photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 52(1):297–314

    Article  CAS  PubMed  Google Scholar 

  • Miyao M, Masumoto C, Miyazawa S, Fukayama H (2011) Lessson from engineering a single- cell C4 photosynthetic pathway in to rice. J Exp Bot 62(11):3021–3029

    Article  CAS  PubMed  Google Scholar 

  • Molla KA, Karmakar S, Chanda PK, Ghosh S, Sarkar SN, Datta SK, Datta K (2013) Rice oxalate oxidase gene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice. Mol Plant Pathol 14(9):910–922

    Article  CAS  PubMed  Google Scholar 

  • Oukarroum A, Strasser RJ, Schansker G (2012) Heat stress and the photosynthetic electron transport chain of the lichen Parmelina tiliacea (Hoffm.) Ach. in the dry and the wet state: differences and similarities with the heat stress response of higher plants. Photosynth Res 111(3):303–314

    Article  CAS  PubMed  Google Scholar 

  • Robaina-Este´vez S, Nikoloski Z (2016) Metabolic Network Constrains Gene Regulation of C4 Photosynthesis: the Case of Maize. Plant Cell Physiol 57(5):933–943

    Article  Google Scholar 

  • Roth-Nebelsick A, Uhl D, Mosbrugger V, Kerp H (2001) Evolution and function of leaf venation architecture: a review. Ann Bot 87(5):553–566

    Article  Google Scholar 

  • Sage RF (2004) The evolution of C4 photosynthesis. New Phytol 161(2):341–370

    Article  CAS  Google Scholar 

  • Strasser A, Tsimilli-Michael M, Srivastava A (2004) Analysis of the fluorescence transient. In: Papageorgiou GC, Govindjee (eds) Chlorophyll fluorescence: a signature of photosynthesis. Advances in photosynthesis and respiration series (Govindjee,Series ed), vol 19. Springer, Dordrecht, pp 321–362

    Chapter  Google Scholar 

  • Taniguchi Y, Ohkawa H, Masumoto C, Fukuda T, Tamai T, Lee K, Sudoh S, Tsuchida H, Sasaki H, Fukayama H, Miyao M (2008) Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice. J Exp Bot 59(7):1799–1809

    Article  CAS  PubMed  Google Scholar 

  • Tilman D, Socolow R, Foley JA (2009) Beneficial biofuels-the food, energy, and environment trilemma. Science 325(5938):270–271

    Article  CAS  PubMed  Google Scholar 

  • Tolley BJ, Sage TL, Langdale JA, Hibberd JM (2012) Individual maize chromosomes in the C3 plant oat can increase bundle sheath cell size and vein density. Plant Physiol 159(4):1418–1427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsuchida H, Tamai T, Fukayama H (2001) High level expression of C4-specific NADP-malic enzyme in leaves and impairment of photoautotrophic growth of C3 plant rice. Plant Cell Physiol 42(2):138–145

    Article  CAS  PubMed  Google Scholar 

  • Ueno O, Kawano Y, Wakayama M, Takeda T (2006) Leaf vascular systems in C3 and C4 grasses: a two-dimensional analysis. Ann Bot 97(4):611–621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Heerden PDR, Strasser RJ, Kru¨ger GHJ (2004) Reduction of dark chilling stress in N2-fixing soybean by nitrate as indicated by chlorophyll a fluorescence kinetics. Physiol Plant 121(2): 239–249.

    Article  PubMed  Google Scholar 

  • Wang Q, Lu CM, Zhang QD (2002) Characterization of photosynthesis, photoinhibition and the activities of C4pathway enzymes in a superhigh-yield rice, Liangyoupeijiu. Sci China Life Sci 45(5):468–476

    Article  CAS  Google Scholar 

  • Wang DZ, Chi W, Wang SH, Jiao DM, Wu S, Li X, Li CQ, Zhang YH, Luo YC (2004) Characteristics of transgenic rice overexpression maize photosynthetic enzymes for breeding two-line hybrid rice. Acta Agron Sin 30(3):248–252

    CAS  Google Scholar 

  • Xu Z, Zhou G (2008) Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. J Exp Bot 59(12):3317–3325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao GQ, Ma BL, Ren CZ (2007) Growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. Crop Sci 47(1):123–131

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are thankful to Prof. K.C Bansal (NBPGR, New Delhi) for providing us the C4 genes. We are grateful to Prof. Arun Lahiri Majumdar (Bose Institute, Kolkata) for providing instrument used to calculate photosynthetic efficiency and for his valuable suggestion. Special thanks to Department of Biotechnology (DBT) Government of India (Project sanction number: BT/01/COE/06/05) and ICAR (Project sanction number: Cs 11/7/2014) for funding this project.

Author contributions

PS designed and performed all the experiments, wrote manuscript, prepared figures and tables. SG helped in genomic DNA isolation and southern hybridization. SS and PC guided thoroughly in experiment. AM helped in calculating photosynthetic efficiency. SKD and KD designed and supervised the experiments and revised and finalized the manuscript.

Author information

Authors and Affiliations

  1. Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

    Priyanka Sen, Subhrajyoti Ghosh, Sailendra Nath Sarkar, Palash Chanda, Swapan Kumar Datta & Karabi Datta

  2. Visva-Bharati University, Santiniketan, 731235, West Bengal, India

    Swapan Kumar Datta

  3. Centre for Diabetes Research, The Methodist Hospital Research Institute, 6670 Bertner, Houston, TX, 77030, USA

    Palash Chanda

  4. Division of Plant Biology, Bose Institute, Kolkata, 700054, India

    Abhishek Mukherjee

Authors
  1. Priyanka Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Subhrajyoti Ghosh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sailendra Nath Sarkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Palash Chanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Abhishek Mukherjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Swapan Kumar Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karabi Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karabi Datta.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11240_2016_1094_MOESM1_ESM.tif

Vector Construct used for transformation by biolistic method. (a) Vector construct showing CA and PEPC gene along with green tissue specific promoters. (b) PPDK vector construct with its own promoter and terminator (TIF 569 KB)

Showing melting curve for all three genes obtained from real time PCR; (a) CA; (b) PEPC; (c) PPDK (TIF 10545 KB)

Pipeline plot demonstrating phenomenological yield model (TIF 2831 KB)

Supplementary Table 1 (DOCX 13 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sen, P., Ghosh, S., Sarkar, S.N. et al. Pyramiding of three C4 specific genes towards yield enhancement in rice. Plant Cell Tiss Organ Cult 128, 145–160 (2017). https://doi.org/10.1007/s11240-016-1094-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-016-1094-2

Keywords

Search

Navigation