Sample Preparation of Arabidopsis thaliana Shoot Apices for Expression Studies of Photoperiod-Induced Genes

  • Fernando Andrés
  • Stefano Torti
  • Coral Vincent
  • George CouplandEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)


Plants produce new organs from a population of pluripotent cells which are located in specific tissues called meristems. One of these meristems, the shoot apical meristem (SAM), gives rise to leaves during the vegetative phase and flowers during the reproductive phase. The transition from vegetative SAM to an inflorescence meristem (IM) is a dramatic developmental switch, which has been particularly well studied in the model species Arabidopsis thaliana. This developmental switch is controlled by multiple environmental signals such as day length (or photoperiod), and it is accompanied by changes in expression of hundreds of genes. A major interest in plant biology is to identify and characterize those genes which are regulated in the stem cells of the SAM in response to the photoperiodic signals. In this sense, techniques such as RNA in situ hybridization (RNA ISH) have been very successfully employed to detect the temporal and spatial expression patterns of genes in the SAM. This method can be specifically optimized for photoperiodic-flowering studies. In this chapter, we describe improved methods to generate plant material and histological samples to be combined with RNA ISH in flowering-related studies.

Key words

Flowering Photoperiod Shoot apical meristem Gene expression studies In situ hybridization Arabidopsis thaliana 



The work in George Coupland’s lab is funded by the European Research Council, the Cluster of Excellence in Plant Sciences (CEPLAS), and a core grant from the Max Planck Society.


  1. 1.
    Koornneef M, Hanhart CJ, van der Veen JH (1991) A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol Gen Genet 229(1):57–66CrossRefPubMedGoogle Scholar
  2. 2.
    Andres F, Coupland G (2012) The genetic basis of flowering responses to seasonal cues. Nat Rev Genet 13(9):627–639CrossRefPubMedGoogle Scholar
  3. 3.
    Blazquez MA, Soowal LN, Lee I, Weigel D (1997) LEAFY expression and flower initiation in Arabidopsis. Development 124(19):3835–3844PubMedGoogle Scholar
  4. 4.
    Mantegazza O, Gregis V, Chiara M, Selva C, Leo G, Horner DS, Kater MM (2014) Gene coexpression patterns during early development of the native Arabidopsis reproductive meristem: novel candidate developmental regulators and patterns of functional redundancy. Plant J 79(5):861–877CrossRefPubMedGoogle Scholar
  5. 5.
    Schmid M, Uhlenhaut NH, Godard F, Demar M, Bressan R, Weigel D, Lohmann JU (2003) Dissection of floral induction pathways using global expression analysis. Development 130(24):6001–6012CrossRefPubMedGoogle Scholar
  6. 6.
    Torti S, Fornara F, Vincent C, Andres F, Nordstrom K, Gobel U, Knoll D, Schoof H, Coupland G (2012) Analysis of the Arabidopsis shoot meristem transcriptome during floral transition identifies distinct regulatory patterns and a leucine-rich repeat protein that promotes flowering. Plant Cell 24(2):444–462PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Jackson D (ed) (1992) In sit hybridization in plants. Molecular plant pathology. A practical approach. Oxford University Press, OxfordGoogle Scholar
  8. 8.
    Carles CC, Ha CM, Jun JH, Fiume E, Fletcher JC (2010) Analyzing shoot apical meristem development. Plant Dev Biol 655:105–129CrossRefGoogle Scholar
  9. 9.
    Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63(2):378–383PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Hejatko J, Blilou I, Brewer PB, Friml J, Scheres B, Benkova E (2006) In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples. Nat Protoc 1(4):1939–1946CrossRefPubMedGoogle Scholar
  11. 11.
    Drea S, Derbyshire P, Koumproglou R, Dolan L, Doonan JH, Shaw P (2009) In situ analysis of gene expression in plants. Methods Mol Biol 513:229–242CrossRefPubMedGoogle Scholar
  12. 12.
    Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 59:573–594CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Fernando Andrés
    • 1
  • Stefano Torti
    • 1
  • Coral Vincent
    • 1
  • George Coupland
    • 1
    Email author
  1. 1.Max Planck Institute for Plant Breeding ResearchCologneGermany

Personalised recommendations