A correlation between leaf shape and its related key genes in Viola albida complex

  • Krishnamoorthy Srikanth
  • Robert S. Hill
  • Sung Soo WhangEmail author
Plant Tissue Culture


Simple to compound leaves occur in the Viola albida complex, which comprises the simple, finely serrate leaves of V. albida Palib., the deeply lobed leaves of V. albida var. takahashii (Nakai) Nakai, and the compound leaves of Viola chaerophylloides (Regel) W. Becker. To identify a correlation between the different leaf forms and the expression of several key genes with roles in leaf morphogenesis, the distinct leaf forms occurring within these species were generated by tissue culture of the V. chaerophylloides petiole, for comparison with wild-type leaves. Compound leaves were generally formed from a petiole explant taken close to the leaf blade, whereas simple leaves resulted from petiole explants taken close to the petiole base. KNOTTED-1 (VaKN1), SHOOTMERISTEMLESS (VaSTM), CUP-SHAPED COTYLEDON-2 (VaCUC2), and ASYMMETRIC LEAVES 1 (VaAS1), which are known to play key roles during compound leaf patterning and morphogenesis, were isolated and multiple sequence alignment revealed that there was no sequence variation at the amino acid level within each gene and between the three varieties. Phylogenetic analysis confirmed that the isolated genes were homologous to KN, STM, CUC2, and AS1. The expression of VaKN1, VaSTM, and VaCUC2 was significantly elevated in the in vitro-cultured deeply lobed and compound leaves, as well as in V. chaerophylloides and V. albida var. takahashii plants, but was very low in the in vitro-cultured simple leaves and V. albida plants. These findings demonstrated that elevated transcripts of VaKN1, VaSTM, and VaCUC2 lead to the development of compound and deeply lobed leaves in the V. albida complex.


Differential gene expression Leaf shape variation Petiole culture Leaf morphology candidate genes VaKN1 VaSTM VaCUC2 VaAS1 



This paper was partly supported by “Research Base Construction Fund Support Program” funded by Chonbuk National University in 2016. The authors would like to thank the reviewers and the handling editor for helping us improve the quality of the manuscript.

Supplementary material

11627_2019_9975_MOESM1_ESM.docx (24 kb)
ESM 1 (DOCX 24 kb)


  1. Bar M, Ori N (2014) Leaf development and morphogenesis. Development 141:4219–4230Google Scholar
  2. Bharathan G, Goliber TE, Moore C, Kessler S, Pham T, Sinha NR (2002) Homologies in leaf form inferred from KNOXI gene expression during development. Science 296:1858–1860CrossRefPubMedGoogle Scholar
  3. Champagne C, Sinha N (2004) Compound leaves: equal to the sum of their parts? Development 131:4401–4412CrossRefPubMedGoogle Scholar
  4. Chitwood DH, Ranjan A, Kumar R, Ichihashi Y, Zumstein K, Headland LR, Ostria-Gallardo E, Aguilar-Martinez JA, Bush S, Carriedo L, Fulop D, Martinez CC, Peng J, Maloof JN, Sinha NR (2014) Resolving distinct genetic regulators of tomato leaf shape within a heteroblastic and ontogenetic context. Plant Cell 26:3616–3629CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  6. Chuck G, Lincoln C, Hake S (1996) KNAT1 induces lobed leaves with ectopic meristems when over expressed in Arabidopsis. Plant Cell 8:1277–1289PubMedPubMedCentralGoogle Scholar
  7. Cronquist A (1988) The evolution and classification of flowering plants, 2nd ed. The New York Botanical Garden, Bronx, NYGoogle Scholar
  8. Dkhar J, Pareek A (2014) What determines a leaf’s shape? EvoDevo 5:1–19Google Scholar
  9. Doyle JA, Endress PK (2000) Morphological phylogenetic analysis of basal angiosperms: comparison and combination with molecular data. Int J Plant Sci 161:S121–S153CrossRefGoogle Scholar
  10. Efroni I, Eshed Y, Lifschitz E (2010) Morphogenesis of simple and compound leaves: a critical review. Plant Cell 22:1019–1032CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hareven D, Gutfinger T, Parnis A, Eshed Y, Lifschitz E (1996) The making of a compound leaf: genetic manipulation of leaf architecture in tomato. Cell 84:735–744CrossRefPubMedGoogle Scholar
  12. Hay A, Tsiantis M (2006) The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsuta. Nat Genet 38:942–947CrossRefPubMedGoogle Scholar
  13. Hay A, Tsiantis M (2010) KNOX genes: versatile regulators of plant development and diversity. Development 137:3153–3165CrossRefPubMedGoogle Scholar
  14. Janssen BJ, Lund L, Sinha N (1998) Overexpression of a homeobox gene LeT6 reveals indeterminate features in the tomato compound leaf. Plant Physiol 117:771–786CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kim GT, Um TW (1995) Effects of gibberellic acid treatment on germination in a study for the utilization of wild herbaceous species. Kor J Environ Ecol 9: 56–61Google Scholar
  16. Kim KS (1986) Studies of comparative morphology on the Korean Viola species. Ph. D thesis. Sung Kyun Kwan University, Republic of Korea (
  17. Kim KS, Sun BY, Whang SS, Chung GH (1991) Biosystematic study on the genus Viola in Korea – comparative morphology of the Viola albida complex. Korean J Bot 34:229–238Google Scholar
  18. Kim M, McCormick S, Timmermans M, Sinha N (2003) The expression domain of PHANTASTICA determines leaflet placement in compound leaves. Nature 425:102CrossRefGoogle Scholar
  19. Ko MK, Yang J, Jin YH, Lee CH, Oh BJ (1998) Genetic relationships of Viola species evaluated by random polymorphic DNA analysis. J Hortic Sci Biotechnol 73:601–605CrossRefGoogle Scholar
  20. Kumar P, Elsaidi HR, Zorniak B, Laurens E, Yang J, Bacchu V, Wang M, Wiebe LI (2016). Synthesis and Biological Evaluation of Iodoglucoazomycin (I‐GAZ), an Azomycin–Glucose Adduct with Putative Applications in Diagnostic Imaging and Radiotherapy of Hypoxic Tumors. Chem Med Chem 11(15):1638–45.Google Scholar
  21. Lee CH, Han NY (1994) Effect of cold treatment on seed germination in Viola species native to Korea. J Kor Soc Hort Sci 12:342–343Google Scholar
  22. Lincoln C, Long J, Yamaguchi J, Serikawa K, Hake S (1994) A KNOTTED1-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants. Plant Cell 6:1859–1876PubMedPubMedCentralGoogle Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. Piazza P, Bailey CD, Cartolano M, Krieger J, Cao J, Ossowski S, Schneeberger K, He F, de Meaux J, Hall N, MacLeod N, Filatov D, Hay A, Tsiantis M (2010) Arabidopsis thaliana leaf form evolved via loss of KNOX expression in leaves in association with selective sweep. Curr Biol 20:2223–2228CrossRefPubMedGoogle Scholar
  25. Pinto FL, Lindblad D (2010) A guide for in-house design of template-switch-based 5′ rapid amplification of cDNA and systems. Anal Biochem 397:222–232CrossRefGoogle Scholar
  26. Rast-Somssich MI, Broholm S, Jenkins H, Canales C, Vlad V, Kwantes M, Bilsborough G, DelloIoio R, Ewing RM, Laufs P, Huijser P, Ohno C, Heisler MG, Hay A, Tsiantis M (2016) Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta. Genes Dev 29:2391–2404CrossRefGoogle Scholar
  27. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467CrossRefPubMedPubMedCentralGoogle Scholar
  28. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C T method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  29. Shani E, Burko Y, Ben-Yaakov L, Berger Y, Amsellem Z, Goldshmidt A, Sharon E, Ori N (2009) Stage-specific regulation of Solanum lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX proteins. Plant Cell 21:3078–3092CrossRefPubMedPubMedCentralGoogle Scholar
  30. Soh H, Auh C, Soh WY, Han K, Kim D, Lee S, Rhee Y (2011) Gene expression changes in Arabidopsis seedlings during short to long term exposure to 3-D clinorotation. Planta 234:255–270CrossRefPubMedGoogle Scholar
  31. Taylor DW, Hickey LJ (1996) Evidence for and implications of an herbaceous origin of angiosperms. In: Taylor DW, Hickey LJ (eds) Flowering plant origin, evolution and phylogeny. Chapman & Hall, New York, pp 232–266CrossRefGoogle Scholar
  32. Tsukaya H (2005) Leaf shape: genetic controls and environmental factors. Intl J Dev Biol 49:547–555Google Scholar
  33. Waites R, Hudson A (1995) PHANTASTICA: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121:2143–2154Google Scholar
  34. Whang SS (2006) Analysis of ITS DNA sequences of the Viola albida complex. Korean J Plant Res 19:628–633Google Scholar
  35. Yoo KO, Jang SK (2010) Infrageneric relationships of Korean Viola based on eight chloroplast markers. J Syst Evol 48:474–481CrossRefGoogle Scholar
  36. Zimmermann W (1952) Main results of the ‘telome theory’. Paleobotanist 1:456–470Google Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Division of Science EducationChonbuk National UniversityJeonjuRepublic of Korea
  2. 2.School of Biological SciencesUniversity of AdelaideAdelaideAustralia

Personalised recommendations