Applied Biochemistry and Biotechnology

, Volume 187, Issue 1, pp 221–238 | Cite as

Genome-Wide Identification and Analysis of Biotic and Abiotic Stress Regulation of C4 Photosynthetic Pathway Genes in Rice

  • Senthilkumar K. Muthusamy
  • Sangram K. Lenka
  • Amit Katiyar
  • Viswanathan Chinnusamy
  • Ashok K. Singh
  • Kailash C. Bansal


Photosynthetic fixation of CO2 is more efficient in C4 than in C3 plants. Rice is a C3 plant and a potential target for genetic engineering of the C4 pathway. It is known that genes encoding C4 enzymes are present in C3 plants. However, no systematic analysis has been conducted to determine if these C4 gene family members are expressed in diverse rice genotypes. In this study, we identified 15 genes belonging to the five C4 gene families in rice genome through BLAST search using known maize C4 photosynthetic pathway genes. Phylogenetic relationship of rice C4 photosynthetic pathway genes and their isoforms with other grass genomes (Brachypodium, maize, Sorghum and Setaria), showed that these genes were highly conserved across grass genomes. Spatiotemporal, hormone, and abiotic stress specific expression pattern of the identified genes revealed constitutive as well as inductive responses of the C4 photosynthetic pathway in different tissues and developmental stages of rice. Expression levels of C4 specific gene family members in flag leaf during tillering stage were quantitatively analyzed in five rice genotypes covering three species, viz. Oryza sativa, ssp. japonica (cv. Nipponbare), Oryza sativa, ssp. indica (cv IR64, Swarna), and two wild species Oryza barthii and Oryza australiensis. The results showed that all the identified genes expressed in rice and exhibited differential expression pattern during different growth stages, and in response to biotic and abiotic stress conditions and hormone treatments. Our study concludes that C4 photosynthetic pathway genes present in rice play a crucial role in stress regulation and might act as targets for C4 pathway engineering via CRISPR-mediated breeding.


C4 rice Carbonic anhydrase (CA) Phosphoenol pyruvate carboxylase (PEPC) Malate dehydrogenase (MDH)  Malic enzyme (ME) and Pyruvate Orthophosphate dikinase (PPDK) 







bundle sheath cells




coding DNA sequence




gibberellic acid


indole-3-acetic acid


mesophyll cell


malate dehydrogenase


massively parallel signature sequencing


1-naphthaleneacetic acid


malic enzyme








phosphoenol pyruvate carboxylase


pyruvate orthophosphate dikinase


ribulosebishosphate carboxylase/oxygenase


radiation use efficiencies



This work was supported by the Indian Council of Agricultural Research (ICAR)-sponsored National Agricultural Innovation Project (NAIP) project. SKM gratefully acknowledges the Indian Agricultural Research Institute (IARI) for IARI-Junior Research Fellowship grant and Department of Science and Technology (DST), Government of India for DST-INSPIRE fellowship grant. The authors acknowledge National Phytotron Facility, IARI, for providing space for growing plants.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. 1.
    Hibberd, J. M., Sheehy, J. E., & Langdale, J. A. (2008). Using C4 photosynthesis to increase the yield of rice-rationale and feasibility. Current Opinion in Plant Biology, 11(2), 228–231. Scholar
  2. 2.
    Elert, E. (2014). Rice by the numbers: A good grain. Nature, 514(7524), S50–S51. Scholar
  3. 3.
    Lobell, D. B., Schlenker, W., & Costa-Roberts, J. (2011). Climate trends and global crop production since 1980. Science, 333(6042), 616–620. (New York, N.Y.).Google Scholar
  4. 4.
    Welch, J. R., Vincent, J. R., Auffhammer, M., Moya, P. F., Dobermann, A., & Dawe, D. (2010). Rice yields in tropical/subtropical Asia exhibit large but opposing sensitivities to minimum and maximum temperatures. Proceedings of the National Academy of Sciences of the United States of America, 107(33), 14562–14567. Scholar
  5. 5.
    Bansal, K. C., Viret, J. F., Haley, J., Khan, B. M., Schantz, R., & Bogorad, L. (1992). Transient expression from cab-m1 and rbcS-m3 promoter sequences is different in mesophyll and bundle sheath cells in maize leaves. Proceedings of the National Academy of Sciences of the United States of America, 89(8), 3654–3658. Scholar
  6. 6.
    Bansal, K. C., & Bogorad, L. (1993). Cell type-preferred expression of maize cab-m1: Repression in bundle sheath cells and enhancement in mesophyll cells. Proceedings of the National Academy of Sciences of the United States of America, 90(9), 4057–4061. Scholar
  7. 7.
    Sheehy, J. E., Ferrer, A. B., Mitchell, P. L., Pablico, P., & Dionora, M. J. A. (2008). How the rice crop works and why it needs a new engine. In Charting New Pathways to C4 Rice pp. 3–26. doi:
  8. 8.
    Sage, R. F., Christin, P. A., & Edwards, E. J. (2011). The C4 plant lineages of planet Earth. Journal of Experimental Botany, 62(9), 3155–3169. Scholar
  9. 9.
    Ludwig, M. (2013). Evolution of the C4 photosynthetic pathway: Events at the cellular and molecular levels. Photosynthesis Research, 117(1–3), 147–161. Scholar
  10. 10.
    Kiniry, J. R. R., Jones, C. A. A., O’toole, J. C. C., Blanchet, R., Cabelguenne, M., & Spanel, D. A. A. (1989). Radiation-use efficiency in biomass accumulation prior to grain-filling for five grain-crop species. Field Crops Research, 20(1), 51–64. Scholar
  11. 11.
    Mitchell, P. L., & Sheehy, J. E. (2006). Supercharging rice photosynthesis to increase yield. New Phytologist, 171(4), 688–693. Scholar
  12. 12.
    Long, S. P., Zhu, X.-G. G., Naidu, S. L., & Ort, D. R. (2006). Can improvement in photosynthesis increase crop yields? Plant, Cell & Environment, 29(3), 315–330. Scholar
  13. 13.
    Kajala, K., Covshoff, S., Karki, S., Woodfield, H., Tolley, B. J., Dionora, M. J. A., Mogul, R. T., Mabilangan, A. E., Danila, F. R., Hibberd, J. M., & Quick, W. P. (2011). Strategies for engineering a two-celled C(4) photosynthetic pathway into rice. Journal of Experimental Botany, 62(9), 3001–3010. Scholar
  14. 14.
    Miyao, M., Masumoto, C., Miyazawa, S. I., & Fukayama, H. (2011). Lessons from engineering a single-cell C4 photosynthetic pathway into rice. Journal of Experimental Botany, 62(9), 3021–3029. Scholar
  15. 15.
    Langdale, J. A. (2011). C4 cycles: Past, present, and future research on C4 photosynthesis. Plant Cell, 23(11), 3879–3892. Scholar
  16. 16.
    von Caemmerer, S., Quick, W. P., & Furbank, R. T. (2012). The development of C4 rice: Current progress and future challenges. Science, 336(6089), 1671–1672. Scholar
  17. 17.
    Hibberd, J. M., & Quick, W. P. (2002). Characteristics of C4 photosynthesis in stems and petioles of C3 flowering plants. Nature, 415(6870), 451–454. Scholar
  18. 18.
    Muthusamy, S. K., Singh, S. K., Singh, I., Singh, A. K., Chinnusamy, V., & Bansal, K. C. (2013). Expression analysis of C4 photosynthetic pathway related genes in seven rice genotypes during grain filling stage. Proc Indian Natl Sci Acad, 79(1), 91–98.Google Scholar
  19. 19.
    Imaizumi, N., Samejima, M., & Ishihara, K. (1997). Characteristics of photosynthetic carbon metabolism of spikelets in rice. Photosynthesis Research, 52(2), 75–82. Scholar
  20. 20.
    Rangan, P., Furtado, A., & Henry, R. J. (2016). New evidence for grain specific C4 photosynthesis in wheat. Scientific Reports, 6(1), 31721. Scholar
  21. 21.
    Langdale, J. A., & Nelson, T. (1991). Spatial regulation of photosynthetic development in C4 plants. Trends Genet: TIG, 7(6), 191–196. Scholar
  22. 22.
    Sage, R. F. (2004). The evolution of C4 photosynthesis. New Phytol, 161(2), 341–370. Scholar
  23. 23.
    Makino, A., Sakuma, I., Sudo, E., & Mae, T. (2003). Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation. Plant and Cell Physiology, 44(9), 952–956. Scholar
  24. 24.
    Raines, C. A. (2011). Increasing photosynthetic carbon assimilation in C3 plants to improve crop yield: Current and future strategies. Plant Physiology, 155(1), 36–42. Scholar
  25. 25.
    Matsuoka, M., Furbank, R. T., Fukayama, H., & Miyao, M. (2001). Molecular engineering of C4 photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology, 52(1), 297–314. Scholar
  26. 26.
    Emanuelsson, O., Brunak, S., von Heijne, G., & Nielsen, H. (2007). Locating proteins in the cell using TargetP, SignalP and related tools. Nature Protocols, 2(4), 953–971. Scholar
  27. 27.
    Horton, P., Park, K.-J. J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C. J., & Nakai, K. (2007). WoLF PSORT: protein localization predictor. Nucleic Acids Research, 35(Web Server issue), W585–W587. Scholar
  28. 28.
    Goodstein, D. M., Shu, S., Howson, R., Neupane, R., Hayes, R. D., Fazo, J., Mitros, T., Dirks, W., Hellsten, U., Putnam, N., & Rokhsar, D. S. (2012). Phytozome: A comparative platform for green plant genomics. Nucleic Acids Research, 40(Database issue), D1178–D1186. Scholar
  29. 29.
    Edgar, R. C. (2004). MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), 1792–1797. Scholar
  30. 30.
    Letunic, I., Doerks, T., & Bork, P. (2012). SMART 7: Recent updates to the protein domain annotation resource. Nucleic Acids Research, 40(Database issue), D302–D305. Scholar
  31. 31.
    Muthusamy, S. K., Dalal, M., Chinnusamy, V., & Bansal, K. C. (2016). Differential regulation of genes coding for organelle and cytosolic ClpATPases under biotic and abiotic stresses in wheat. Frontiers in Plant Science, 7, 929. Scholar
  32. 32.
    Katiyar, A., Smita, S., Muthusamy, S. K., Chinnusamy, V., Pandey, D. M., & Bansal, K. C. (2015). Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis. Frontiers in Plant Science, 6, 506. Scholar
  33. 33.
    Hruz, T., Laule, O., Szabo, G., Wessendorp, F., Bleuler, S., Oertle, L., et al. (2008). Genevestigator v3: A reference expression database for the meta-analysis of transcriptomes. Advances in Bioinformatics, 2008, 420747–420745. Scholar
  34. 34.
    Muthusamy, S. K., Dalal, M., Chinnusamy, V., & Bansal, K. C. (2017). Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat. Journal of Plant Physiology, 211, 100–113. Scholar
  35. 35.
    Nakano, M., Nobuta, K., Vemaraju, K., Tej, S. S., Skogen, J. W., & Meyers, B. C. (2006). Plant MPSS databases: Signature-based transcriptional resources for analyses of mRNA and small RNA. Nucleic Acids Research, 34(90001), D731–D735. Scholar
  36. 36.
    Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods, 25(4), 402–408. (San Diego, Calif.).Google Scholar
  37. 37.
    Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731–2739. Scholar
  38. 38.
    Price, G. D., von Caemmerer, S., Evans, J. R., Yu, J. W., Lloyd, J., Oja, V., Kell, P., Harrison, K., Gallagher, A., & Badger, M. R. (1994). Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthetic CO2 assimilation. Planta, 193(3), 331–340. Scholar
  39. 39.
    DiMario, R. J., Clayton, H., Mukherjee, A., Ludwig, M., & Moroney, J. V. (2017). Plant carbonic anhydrases: Structures, locations, evolution, and physiological roles. Molecular Plant, 10(1), 30–46. Scholar
  40. 40.
    Moroney, J. V., Bartlett, S. G., & Samuelsson, G. (2001). Carbonic anhydrases in plants and algae: Invited review. Plant, Cell and Environment, 24(2), 141–153. Scholar
  41. 41.
    Wang, X., Gowik, U., Tang, H., Bowers, J., Westhoff, P., & Paterson, A. (2009). Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses. Genome Biology, 10(6), R68. Scholar
  42. 42.
    Vidal, J., & Chollet, R. (1997). Regulatory phosphorylation of C4 PEP carboxylase. Trends in Plant Science, 2(6), 230–237. Scholar
  43. 43.
    Masumoto, C., Miyazawa, S.-I. S.-I., Ohkawa, H., Fukuda, T., Taniguchi, Y., Murayama, S., et al. (2010). Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation. Proceedings of the National Academy of Sciences, 107(11), 5226–5231. Scholar
  44. 44.
    Chollet, R., Vidal, J., & O’Leary, M. H. (1996). Pyruvate carboxylase: A ubiquitous, highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47(1), 273–298. Scholar
  45. 45.
    Sánchez, R., & Cejudo, F. (2003). Identification and expression analysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice. Plant Physiology, 132(June), 949–957. Scholar
  46. 46.
    Doubnerová, V., & Ryšlavá, H. (2011). What can enzymes of C4 photosynthesis do for C3 plants under stress? Plant Science, 180(4), 575–583. Scholar
  47. 47.
    Chi, W., Yang, J., Wu, N., & Zhang, F. (2004). Four rice genes encoding NADP malic enzyme exhibit distinct expression profiles. Bioscience, Biotechnology, and Biochemistry, 68(9), 1865–1874. Scholar
  48. 48.
    Cairoll, L. J., Dunaway-Mariano, D., Smith, C. M., & Chollet, R. (1990). Determination of the catalytic pathway of C4-leaf pyruvate, orthophosphate dikinase from maize. FEBS Letters, 274(1–2), 178–180. Scholar
  49. 49.
    Parsley, K., & Hibberd, J. M. (2006). The Arabidopsis PPDK gene is transcribed from two promoters to produce differentially expressed transcripts responsible for cytosolic and plastidic proteins. Plant Molecular Biology, 62(3), 339–349. Scholar
  50. 50.
    Dong, X., Li, Y., Chao, Q., Shen, J., Gong, X., Zhao, B., & Wang, B. (2016). Analysis of gene expression and histone modification between C4 and non-C4 homologous genes of PPDK and PCK in maize. Photosynthesis Research, 129(1), 71–83. Scholar
  51. 51.
    Hibberd, J. M., & Covshoff, S. (2010). The regulation of gene expression required for C4 photosynthesis. Annual Review of Plant Biology, 61(1), 181–207. Scholar
  52. 52.
    Aubry, S., Brown, N. J., & Hibberd, J. M. (2011). The role of proteins in C3 plants prior to their recruitment into the C4 pathway. Journal of Experimental Botany, 62(9), 3049–3059. Scholar
  53. 53.
    Wang, Q., Zhang, Q., Fan, D., & Lu, C. (2006). Photosynthetic light and CO2 utilization and C4 traits of two novel super-rice hybrids. Journal of Plant Physiology, 163(5), 529–537. Scholar
  54. 54.
    Wang, P., Vlad, D., & Langdale, J. A. (2016). Finding the genes to build C4 rice. Current Opinion in Plant Biology, 31, 44–50. Scholar
  55. 55.
    Xu, J., Bräutigam, A., Weber, A. P. M., & Zhu, X. G. (2016). Systems analysis of cis-regulatory motifs in C4photosynthesis genes using maize and rice leaf transcriptomic data during a process of de-etiolation. Journal of Experimental Botany, 67(17), 5105–5117. Scholar
  56. 56.
    Chang, T. T. (1976). The origin, evolution, cultivation, dissemination, and diversification of Asian and African rices. Euphytica, 25(1), 425–441. Scholar
  57. 57.
    Khush, G. S. (1997). Origin, dispersal, cultivation and variation of rice. Plant Molecular Biology, 35(1/2), 25–34. Scholar
  58. 58.
    Tsuzuki, M., Miyachi, S., & Edwards, G. E. (1985). Localization of carbonic anhydrase in mesophyll cells of terrestrial C3 plants in relation to CO2 assimilation. Plant and Cell Physiology, 26(5), 881–891. Scholar
  59. 59.
    Rumeau, D., Cuine, S., Fina, L., Gault, N., Nicole, M., & Peltier, G. (1996). Subcellular distribution of carbonic anhydrase in Solanum tuberosum L leaves—Characterization of two compartment-specific isoforms. Planta, 199(1), 79–88. Scholar
  60. 60.
    Yu, S., Zhang, X., Guan, Q., Takano, T., & Liu, S. (2007). Expression of a carbonic anhydrase gene is induced by environmental stresses in rice (Oryza sativa L.). Biotechnology Letters, 29(1), 89–94. Scholar
  61. 61.
    Matsuoka, M., & Hata, S. (1987). Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants. Plant Physiology, 85(4), 947–951.Google Scholar
  62. 62.
    Yeo, M. E., Yeo, A. R., & Flowers, T. J. (1994). Photosynthesis and photorespiration in the genus Oryza. Journal of Experimental Botany, 45(5), 553–560. Scholar
  63. 63.
    Cornic, G., Bukhov, N. G., Wiese, C., Bligny, R., & Heber, U. (2000). Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping. Planta, 210(3), 468–477. Scholar
  64. 64.
    Saccardy, K., Cornic, G., Brulfert, J., & Reyss, A. (1996). Effect of drought stress on net CO2 uptake by Zea leaves. Planta, 199(4), 589–595. Scholar
  65. 65.
    Bandyopadhyay, A., Datta, K., Zhang, J., Yang, W., Raychaudhuri, S., Miyao, M., & Datta, S. K. (2007). Enhanced photosynthesis rate in genetically engineered indica rice expressing pepc gene cloned from maize. Plant Science, 172(6), 1204–1209. Scholar
  66. 66.
    Cheng, Y., & Long, M. (2007). A cytosolic NADP-malic enzyme gene from rice (Oryza sativa L.) confers salt tolerance in transgenic Arabidopsis. Biotechnology Letters, 29(7), 1129–1134. Scholar
  67. 67.
    Brar, D. S., & Khush, G. S. (2006). Cytogenetic manipulation and germplasm enhancement of rice (Oryza sativa L.). In: Singh RJ, Jauhar PP (eds) Genetic resources, chromosome engineering and crop improvement. CRC Press, Boca Raton, FL, 115–158.
  68. 68.
    Liu, S., Cheng, Y., Zhang, X., Guan, Q., Nishiuchi, S., Hase, K., & Takano, T. (2007). Expression of an NADP-malic enzyme gene in rice (Oryza sativa. L) is induced by environmental stresses; over-expression of the gene in Arabidopsis confers salt and osmotic stress tolerance. Plant Molecular Biology, 64(1–2), 49–58. Scholar
  69. 69.
    Moons, A., Valcke, R., & Van Montagu, M. (1998). Low-oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C3 plant. Plant Journal, 15(1), 89–98. Scholar
  70. 70.
    Henry, R. J., Rice, N., Waters, D. L. E., Kasem, S., Ishikawa, R., Hao, Y., Dillon, S., Crayn, D., Wing, R., & Vaughan, D. (2010). Australian Oryza: Utility and conservation. Rice, 3(4), 235–241. Scholar
  71. 71.
    Wang, L., Czedik-Eysenberg, A., Mertz, R. A., Si, Y., Tohge, T., Nunes-Nesi, A., Arrivault, S., Dedow, L. K., Bryant, D. W., Zhou, W., Xu, J., Weissmann, S., Studer, A., Li, P., Zhang, C., LaRue, T., Shao, Y., Ding, Z., Sun, Q., Patel, R. V., Turgeon, R., Zhu, X., Provart, N. J., Mockler, T. C., Fernie, A. R., Stitt, M., Liu, P., & Brutnell, T. P. (2014). Comparative analyses of C4 and C3 photosynthesis in developing leaves of maize and rice. Nature Biotechnology, 32(11), 1158–1165. Scholar
  72. 72.
    Reyna-Llorens, I., Burgess, S. J., Reeves, G., Singh, P., Stevenson, S. R., Williams, B. P., Stanley, S., & Hibberd, J. M. (2018). Ancient duons may underpin spatial patterning of gene expression in C4 leaves. Proceedings of the National Academy of Sciences, 115(8), 1931–1936. Scholar
  73. 73.
    Heimann, L., Horst, I., Perduns, R., Dreesen, B., Offermann, S., & Peterhansel, C. (2013). A common histone modification code on C4 genes in maize and its conservation in Sorghum and Setaria italica. Plant Physiology, 162(1), 456–469. Scholar
  74. 74.
    Reeves, G., Grangé-Guermente, M. J., & Hibberd, J. M. (2017). Regulatory gateways for cell-specific gene expression in C4 leaves with Kranz anatomy. Journal of Experimental Botany, 68(2), 107–116. Scholar

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Authors and Affiliations

  • Senthilkumar K. Muthusamy
    • 1
    • 2
  • Sangram K. Lenka
    • 1
    • 3
  • Amit Katiyar
    • 1
    • 4
  • Viswanathan Chinnusamy
    • 5
  • Ashok K. Singh
    • 6
  • Kailash C. Bansal
    • 1
    • 3
  1. 1.ICAR-National Research Centre on Plant BiotechnologyICAR-Indian Agricultural Research InstituteNew DelhiIndia
  2. 2.Division of Crop ImprovementICAR-Central Tuber Crops Research InstituteThiruvananthapuramIndia
  3. 3.TERI-Deakin Nanobiotechnology CentreGurgaonIndia
  4. 4.ICMR-All India Institute of Medical ScienceNew DelhiIndia
  5. 5.Division of Plant PhysiologyICAR-Indian Agricultural Research InstituteNew DelhiIndia
  6. 6.Division of GeneticsICAR-Indian Agricultural Research InstituteNew DelhiIndia

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