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Cotyledons contribute to plant growth and hybrid vigor in Arabidopsis

  • Li Wang
  • Pei-Chuan Liu
  • Li Min Wu
  • Jiafu Tan
  • W. James Peacock
  • Elizabeth S. Dennis
Original Article


Main conclusion

In hybrids of Arabidopsis, cotyledons influence the amount and proportion of hybrid vigor in total plant growth.

We found Arabidopsis cotyledons are essential for plant growth and in some hybrids for hybrid vigor. In hybrids between C24 and Landsberg erecta (Ler), biomass vigor (heterosis) occurs in the first few days after sowing (DAS), with hybrid cotyledons being larger than those of their parents. C24xLer hybrids are ahead of their parents in activating photosynthesis and auxin pathway genes in cotyledons at 3–4 DAS. “Earliness” is also present in newly emerged C24xLer hybrid leaves. We showed cotyledon removal at 4 DAS caused significant biomass reduction in later growth in hybrids and parental lines. The biomass decrease caused by cotyledon removal can be partially rescued by exogenous sucrose or auxin with different genotypes responding to sucrose and/or auxin differently. Cotyledon removal has different effects on heterosis in different hybrids. After cotyledon removal, in C24xLer hybrids, both growth and heterosis were reduced in similar proportions, but the level of hybrid vigor was reduced as a proportion of growth in C24xColumbia (Col) and ColxLer hybrids. The removal of cotyledons at 4 DAS markedly decreased the level of growth and eliminated the heterotic phenotype of Wassilewskija (Ws)/Ler hybrids. In mutant Ws/Ler hybrids which had a reduced level of photosynthesis in the cotyledons, there was a reduction in plant growth and loss of heterosis. The variation in contribution of cotyledons to heterosis in different hybrids indicates there are multiple pathways to achieve heterotic phenotypes.


Auxin Heterosis Photosynthesis PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) Shi-yo-u 1 (cyo1Snowy cotyledon 2 (sco2) 



Shi-yo-u 1


Day after sowing


Fresh weight






Mid-parent value


1-Naphthaleneacetic acid






Snowy cotyledon 1





We thank Dr. Ken-ichiro Takamiya in Tokyo Institute of Technology and Professor Barry Pogson at the Australia National University for providing cyo1 and sco2 mutant seeds, Aihua Wang and Bjorg Sherman for technical assistance, Neil Smith, Dr. Ian Greaves and Dr. Anyu Zhu for helpful discussion and suggestions on this project, Dr. Ming-Bo Wang, Dr. Masumi Robertson and Dr. TJ Higgins for manuscript reviewing.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

425_2018_3068_MOESM1_ESM.xlsx (26 kb)
Supplementary material 1 (XLSX 26 kb)
425_2018_3068_MOESM2_ESM.pdf (902 kb)
Fig. S1 Cotyledon removal at 7 or 10 DAS had less effect on plant biomass at 15 DAS than removal at 4 DAS. The phenotypes (a) and FWs (b) of 15-day-old parents (C24 and Ler), and two reciprocal hybrids (C24xLer and LerxC24) with cotyledon removed (-Cot) at 4, 7 and 10 DAS. The intact parents and hybrid plants are used as controls (Control). n > 10. Scale bar = 1 cm applies to all images. *indicates significant differences from the MPV at P (Student’s t test) < 0.05. Error bars = SE. Fig. S2 Plants with cotyledon removed had a slower developmental rate than the intact plants. a The flowering time of the parents and hybrids having cotyledons removed (-Cot) at 4 DAS compared to the control plants. b The number of rosette leaves produced at the time of flowering in Ler and Ler having cotyledon removed at 4 DAS. * indicates significant differences at P (Student’s t test) < 0.05 from the control plants. Error bars = SE. n = 15 – 20. Fig. S3 The phenotypes of 15-day-old parents C24 and Ler and hybrids C24xLer growing on media with added sucrose or auxin compared to plants growing on standard media. “Control” indicates intact plants, “-Cot” indicates the plants had cotyledons removed at 4 DAS. Scale bar = 1 cm. Fig. S4 Cotyledon removal at 4 DAS caused biomass loss in a number of hybrid combinations. a Result of cotyledon removal experiment in Fig. 5a (EXP1_May2018) was confirmed in an independent sowing (EXP2_June2018). n = 9-18 per line. b A summary of the level of heterosis in two experiments. The numbers above columns indicate the level of heterosis in the hybrids with/without cotyledons removed. c The FWs of the parents Ws and Ler, and Ws/Ler hybrids with cotyledons removed (-Cot) at 4 DAS compared to the control plants at 19 DAS. * indicates significant differences at P (Student’s t test) < 0.05 from MPV. Error bars = SE. Fig. S5 cyo1 showed phenotypic variation in the colour of cotyledons (lL.G., light green; P, pale; W, white). Photo was taken at 10 DAS. Scale bar = 1 cm. Fig. S6 Loss of heterosis in cyo1/sco2 hybrid at 19 DAS. a The FWs of Ws, Ler, Ws/Ler hybrids, cyo1, sco2 and cyo1/sco2 hybrids at 19 DAS. Mutants were grouped by phenotype category. ** indicates significant differences at P (Student’s t test) < 0.01 from the MPV. Error bars = SE. b Photos of Ws, Ler, Ws/Ler hybrids, cyo1, sco2 and cyo1/sco2 hybrids at 19 DAS. Scale bar = 5 cm (PDF 902 kb)


  1. Albrecht V, Ingenfeld A, Apel K (2006) Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality. Plant Mol Biol 60:507–518. CrossRefPubMedGoogle Scholar
  2. Albrecht V, Ingenfeld A, Apel K (2008) Snowy cotyledon 2: the identification of a zinc finger domain protein essential for chloroplast development in cotyledons but not in true leaves. Plant Mol Biol 66:599–608. CrossRefPubMedGoogle Scholar
  3. Alonso-Peral MM, Trigueros M, Sherman B, Ying H, Taylor JM, Peacock WJ, Dennis ES (2017) Patterns of gene expression in developing embryos of Arabidopsis hybrids. Plant J 89:927–939. CrossRefPubMedGoogle Scholar
  4. Birchler JA, Auger DL, Riddle NC (2003) In search of the molecular basis of heterosis. Plant Cell 15:2236–2239. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Birchler JA, Yao H, Chudalayandi S, Vaiman D, Veitia RA (2010) Heterosis. Plant Cell 22:2105–2112. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bisognin DA, Velasquez L, Widders I (2005) Cucumber seedling dependence on cotyledonary leaves for early growth. Pesqui Agropecu Bras 40:531–539. CrossRefGoogle Scholar
  7. Chandler JW (2008) Cotyledon organogenesis. J Exp Bot 59:2917–2931. CrossRefPubMedGoogle Scholar
  8. Cheng YF, Dai XH, Zhao YD (2007) Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis. Plant Cell 19:2430–2439. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 39:5–17. CrossRefGoogle Scholar
  10. Delker C, Poschl Y, Raschke A, Ullrich K, Ettingshausen S, Hauptmann V, Grosse I, Quint M (2010) Natural variation of transcriptional auxin response networks in Arabidopsis thaliana. Plant Cell 22:2184–2200. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Francisco M, Joseph B, Caligagan H, Li BH, Corwin JA, Lin C, Kerwin R, Burow M, Kliebenstein DJ (2016) The defense metabolite, allyl glucosinolate, modulates Arabidopsis thaliana biomass dependent upon the endogenous glucosinolate pathway. Front Plant Sci 7:774. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fujimoto R, Taylor JM, Shirasawa S, Peacock WJ, Dennis ES (2012) Heterosis of Arabidopsis hybrids between C24 and Col is associated with increased photosynthesis capacity. Proc Natl Acad Sci USA 109:7109–7114. CrossRefPubMedGoogle Scholar
  13. Groszmann M, Greaves IK, Albertyn ZI, Scofield GN, Peacock WJ, Dennis ES (2011) Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic contribution to hybrid vigor. Proc Natl Acad Sci USA 108:2617–2622. CrossRefPubMedGoogle Scholar
  14. Groszmann M, Gonzalez-Bayon R, Greaves IK, Wang L, Huen AK, Peacock WJ, Dennis ES (2014) Intraspecific Arabidopsis hybrids show different patterns of heterosis despite the close relatedness of the parental genomes. Plant Physiol 166:265–280. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Groszmann M, Gonzalez-Bayon R, Lyons RL, Greaves IK, Kazan K, Peacock WJ, Dennis ES (2015) Hormone-regulated defense and stress response networks contribute to heterosis in Arabidopsis F1 hybrids. Proc Natl Acad Sci USA 112:E6397–E6406. CrossRefPubMedGoogle Scholar
  16. Hanley ME, Fegan EL (2007) Timing of cotyledon damage affects growth and flowering in mature plants. Plant Cell Environ 30:812–819. CrossRefPubMedGoogle Scholar
  17. Lilley JLS, Gee CW, Sairanen I, Ljung K, Nemhauser JL (2012) An endogenous carbon-sensing pathway triggers increased auxin flux and hypocotyl elongation. Plant Physiol 160:2261–2270. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Ljung K, Nemhauser JL, Perata P (2015) New mechanistic links between sugar and hormone signalling networks. Curr Opin Plant Biol 25:130–137. CrossRefPubMedGoogle Scholar
  19. Meyer RC, Torjek O, Becher M, Altmann T (2004) Heterosis of biomass production in Arabidopsis. Establishment during early development. Plant Physiol 134:1813–1823. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Penny MG, Moore KG, Lovell PH (1976) The effects of inhibition of cotyledon photosynthesis on seedling development in Cucumis sativus L. Ann Bot 40:815–824. CrossRefGoogle Scholar
  21. Petrasek J, Friml J (2009) Auxin transport routes in plant development. Development 136:2675–2688. CrossRefPubMedGoogle Scholar
  22. Saeki N, Kawanabe T, Ying H, Shimizu M, Kojima M, Abe H, Okazaki K, Kaji M, Taylor JM, Sakakibara H, Peacock WJ, Dennis ES, Fujimoto R (2016) Molecular and cellular characteristics of hybrid vigour in a commercial hybrid of Chinese cabbage. BMC Plant Biol 16:45. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Sarkissian IV, Harris W, Kessinger MA (1964) Differential rates of development of heterotic + nonheterotic young maize seedlings. I. Correlation of differential morphological development with physiological differences in germinating seeds. Proc Natl Acad Sci USA 51:212–218. CrossRefPubMedGoogle Scholar
  24. Shimada H, Mochizuki M, Ogura K, Froehlich JE, Osteryoung KW, Shirano Y, Shibata D, Masuda S, Mori K, Takamiya KI (2007) Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase. Plant Cell 19:3157–3169. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Sun JQ, Qi LL, Li YN, Chu JF, Li CY (2012) PIF4-mediated activation of YUCCA8 expression integrates temperature into the auxin pathway in regulating Arabidopsis hypocotyl growth. PLoS Genet 8:e1002594. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Sun JQ, Qi LL, Li YA, Zhai QZ, Li CY (2013) PIF4 and PIF5 transcription factors link blue light and auxin to regulate the phototropic response in Arabidopsis. Plant Cell 25:2102–2114. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Vanhaeren H, Gonzalez N, Inzé D (2015) A journey through a leaf: phenomics analysis of leaf growth in Arabidopsis thaliana. Arabidopsis Book 13:e0181. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Wang L, Wu LM, Greaves IK, Zhu A, Dennis ES, Peacock WJ (2017) PIF4-controlled auxin pathway contributes to hybrid vigor in Arabidopsis thaliana. Proc Natl Acad Sci USA 114:E3555–E3562. CrossRefPubMedGoogle Scholar
  29. Zagari N, Sandoval-Ibanez O, Sandal N, Su JY, Rodriguez-Concepcion M, Stougaard J, Pribil M, Leister D, Pulido P (2017) SNOWY COTYLEDON 2 promotes chloroplast development and has a role in leaf variegation in both Lotus japonicus and Arabidopsis thaliana. Mol Plant 10:721–734. CrossRefPubMedGoogle Scholar
  30. Zhang HX, Wui Y, Matthew C, Zhou DW, Wang P (2008) Contribution of cotyledons to seedling dry weight and development in Medicago falcata L. N Zeal J Agr Res 51:107–114. CrossRefGoogle Scholar
  31. Zhu AY, Greaves IK, Liu PC, Wu LM, Dennis ES, Peacock WJ (2016) Early changes of gene activity in developing seedlings of Arabidopsis hybrids relative to parents may contribute to hybrid vigour. Plant J 88:597–607. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Li Wang
    • 1
  • Pei-Chuan Liu
    • 1
  • Li Min Wu
    • 2
  • Jiafu Tan
    • 1
  • W. James Peacock
    • 1
    • 2
  • Elizabeth S. Dennis
    • 1
    • 2
  1. 1.Faculty of ScienceUniversity of TechnologySydneyAustralia
  2. 2.Agriculture and Food, Commonwealth Scientific Industrial Research OrganisationCanberraAustralia

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