Skip to main content
PMC home page
The Plant Cell logoLink to The Plant Cell
. 1999 Oct;11(10):1827–1840. doi: 10.1105/tpc.11.10.1827

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

R C Meissner 1, H Jin 1, E Cominelli 1, M Denekamp 1, A Fuertes 1, R Greco 1, H D Kranz 1, S Penfield 1, K Petroni 1, A Urzainqui 1, C Martin 1, J Paz-Ares 1, S Smeekens 1, C Tonelli 1, B Weisshaar 1, E Baumann 1, V Klimyuk 1, S Marillonnet 1, K Patel 1, E Speulman 1, A F Tissier 1, D Bouchez 1, J J Jones 1, A Pereira 1, E Wisman 1
PMCID: PMC144113  PMID: 10521515

Abstract

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Full Text

The Full Text of this article is available as a PDF (167.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aarts M. G., Corzaan P., Stiekema W. J., Pereira A. A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana. Mol Gen Genet. 1995 Jun 10;247(5):555–564. doi: 10.1007/BF00290346. [DOI] [PubMed] [Google Scholar]
  2. Avila J., Nieto C., Cañas L., Benito M. J., Paz-Ares J. Petunia hybrida genes related to the maize regulatory C1 gene and to animal myb proto-oncogenes. Plant J. 1993 Apr;3(4):553–562. doi: 10.1046/j.1365-313x.1993.03040553.x. [DOI] [PubMed] [Google Scholar]
  3. Azpiroz-Leehan R., Feldmann K. A. T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet. 1997 Apr;13(4):152–156. doi: 10.1016/s0168-9525(97)01094-9. [DOI] [PubMed] [Google Scholar]
  4. Ballinger D. G., Benzer S. Targeted gene mutations in Drosophila. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9402–9406. doi: 10.1073/pnas.86.23.9402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baranowskij N., Frohberg C., Prat S., Willmitzer L. A novel DNA binding protein with homology to Myb oncoproteins containing only one repeat can function as a transcriptional activator. EMBO J. 1994 Nov 15;13(22):5383–5392. doi: 10.1002/j.1460-2075.1994.tb06873.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baulcombe D. C. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol Biol. 1996 Oct;32(1-2):79–88. doi: 10.1007/BF00039378. [DOI] [PubMed] [Google Scholar]
  7. Bender J., Fink G. R. A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis. Proc Natl Acad Sci U S A. 1998 May 12;95(10):5655–5660. doi: 10.1073/pnas.95.10.5655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bevan M., Bancroft I., Bent E., Love K., Goodman H., Dean C., Bergkamp R., Dirkse W., Van Staveren M., Stiekema W. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature. 1998 Jan 29;391(6666):485–488. doi: 10.1038/35140. [DOI] [PubMed] [Google Scholar]
  9. Bouchez D., Höfte H. Functional genomics in plants. Plant Physiol. 1998 Nov;118(3):725–732. doi: 10.1104/pp.118.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carroll B. J., Klimyuk V. I., Thomas C. M., Bishop G. J., Harrison K., Scofield S. R., Jones J. D. Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato. Genetics. 1995 Jan;139(1):407–420. doi: 10.1093/genetics/139.1.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Das L., Martienssen R. Site-selected transposon mutagenesis at the hcf106 locus in maize. Plant Cell. 1995 Mar;7(3):287–294. doi: 10.1105/tpc.7.3.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edwards K., Johnstone C., Thompson C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 1991 Mar 25;19(6):1349–1349. doi: 10.1093/nar/19.6.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feldbrügge M., Sprenger M., Hahlbrock K., Weisshaar B. PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit. Plant J. 1997 May;11(5):1079–1093. doi: 10.1046/j.1365-313x.1997.11051079.x. [DOI] [PubMed] [Google Scholar]
  15. Gaymard F., Pilot G., Lacombe B., Bouchez D., Bruneau D., Boucherez J., Michaux-Ferrière N., Thibaud J. B., Sentenac H. Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap. Cell. 1998 Sep 4;94(5):647–655. doi: 10.1016/s0092-8674(00)81606-2. [DOI] [PubMed] [Google Scholar]
  16. Grotewold E., Drummond B. J., Bowen B., Peterson T. The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell. 1994 Feb 11;76(3):543–553. doi: 10.1016/0092-8674(94)90117-1. [DOI] [PubMed] [Google Scholar]
  17. Gubler F., Kalla R., Roberts J. K., Jacobsen J. V. Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI alpha-amylase gene promoter. Plant Cell. 1995 Nov;7(11):1879–1891. doi: 10.1105/tpc.7.11.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirsch R. E., Lewis B. D., Spalding E. P., Sussman M. R. A role for the AKT1 potassium channel in plant nutrition. Science. 1998 May 8;280(5365):918–921. doi: 10.1126/science.280.5365.918. [DOI] [PubMed] [Google Scholar]
  19. Hoeren F. U., Dolferus R., Wu Y., Peacock W. J., Dennis E. S. Evidence for a role for AtMYB2 in the induction of the Arabidopsis alcohol dehydrogenase gene (ADH1) by low oxygen. Genetics. 1998 Jun;149(2):479–490. doi: 10.1093/genetics/149.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Höfgen R., Axelsen K. B., Kannangara C. G., Schüttke I., Pohlenz H. D., Willmitzer L., Grimm B., von Wettstein D. A visible marker for antisense mRNA expression in plants: inhibition of chlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase antisense gene. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1726–1730. doi: 10.1073/pnas.91.5.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Iturriaga G., Leyns L., Villegas A., Gharaibeh R., Salamini F., Bartels D. A family of novel myb-related genes from the resurrection plant Craterostigma plantagineum are specifically expressed in callus and roots in response to ABA or desiccation. Plant Mol Biol. 1996 Nov;32(4):707–716. doi: 10.1007/BF00020211. [DOI] [PubMed] [Google Scholar]
  22. Jackson D., Culianez-Macia F., Prescott A. G., Roberts K., Martin C. Expression patterns of myb genes from Antirrhinum flowers. Plant Cell. 1991 Feb;3(2):115–125. doi: 10.1105/tpc.3.2.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaiser K., Goodwin S. F. "Site-selected" transposon mutagenesis of Drosophila. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1686–1690. doi: 10.1073/pnas.87.5.1686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaneko T., Kotani H., Nakamura Y., Sato S., Asamizu E., Miyajima N., Tabata S. Structural analysis of Arabidopsis thaliana chromosome 5. V. Sequence features of the regions of 1,381,565 bp covered by twenty one physically assigned P1 and TAC clones. DNA Res. 1998 Apr 30;5(2):131–145. doi: 10.1093/dnares/5.2.131. [DOI] [PubMed] [Google Scholar]
  25. Kempin S. A., Liljegren S. J., Block L. M., Rounsley S. D., Yanofsky M. F., Lam E. Targeted disruption in Arabidopsis. Nature. 1997 Oct 23;389(6653):802–803. doi: 10.1038/39770. [DOI] [PubMed] [Google Scholar]
  26. Kirik V., Bäumlein H. A novel leaf-specific myb-related protein with a single binding repeat. Gene. 1996 Dec 12;183(1-2):109–113. doi: 10.1016/s0378-1119(96)00521-5. [DOI] [PubMed] [Google Scholar]
  27. Koes R., Souer E., van Houwelingen A., Mur L., Spelt C., Quattrocchio F., Wing J., Oppedijk B., Ahmed S., Maes T. Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8149–8153. doi: 10.1073/pnas.92.18.8149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Koncz C., Németh K., Rédei G. P., Schell J. T-DNA insertional mutagenesis in Arabidopsis. Plant Mol Biol. 1992 Dec;20(5):963–976. doi: 10.1007/BF00027166. [DOI] [PubMed] [Google Scholar]
  29. Kranz H. D., Denekamp M., Greco R., Jin H., Leyva A., Meissner R. C., Petroni K., Urzainqui A., Bevan M., Martin C. Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J. 1998 Oct;16(2):263–276. doi: 10.1046/j.1365-313x.1998.00278.x. [DOI] [PubMed] [Google Scholar]
  30. Krysan P. J., Young J. C., Tax F., Sussman M. R. Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):8145–8150. doi: 10.1073/pnas.93.15.8145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lüscher B., Eisenman R. N. New light on Myc and Myb. Part II. Myb. Genes Dev. 1990 Dec;4(12B):2235–2241. doi: 10.1101/gad.4.12b.2235. [DOI] [PubMed] [Google Scholar]
  32. Magaraggia F., Solinas G., Valle G., Giovinazzo G., Coraggio I. Maturation and translation mechanisms involved in the expression of a myb gene of rice. Plant Mol Biol. 1997 Dec;35(6):1003–1008. doi: 10.1023/a:1005821518242. [DOI] [PubMed] [Google Scholar]
  33. Martienssen R. A. Functional genomics: probing plant gene function and expression with transposons. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2021–2026. doi: 10.1073/pnas.95.5.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Martin C., Paz-Ares J. MYB transcription factors in plants. Trends Genet. 1997 Feb;13(2):67–73. doi: 10.1016/s0168-9525(96)10049-4. [DOI] [PubMed] [Google Scholar]
  35. McKinney E. C., Ali N., Traut A., Feldmann K. A., Belostotsky D. A., McDowell J. M., Meagher R. B. Sequence-based identification of T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 and act4-1. Plant J. 1995 Oct;8(4):613–622. doi: 10.1046/j.1365-313x.1995.8040613.x. [DOI] [PubMed] [Google Scholar]
  36. Moyano E., Martínez-Garcia J. F., Martin C. Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in antirrhinum flowers. Plant Cell. 1996 Sep;8(9):1519–1532. doi: 10.1105/tpc.8.9.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Müller A., Guan C., Gälweiler L., Tänzler P., Huijser P., Marchant A., Parry G., Bennett M., Wisman E., Palme K. AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J. 1998 Dec 1;17(23):6903–6911. doi: 10.1093/emboj/17.23.6903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nacry P., Camilleri C., Courtial B., Caboche M., Bouchez D. Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics. 1998 Jun;149(2):641–650. doi: 10.1093/genetics/149.2.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Noda K., Glover B. J., Linstead P., Martin C. Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature. 1994 Jun 23;369(6482):661–664. doi: 10.1038/369661a0. [DOI] [PubMed] [Google Scholar]
  40. Oppenheimer D. G., Herman P. L., Sivakumaran S., Esch J., Marks M. D. A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell. 1991 Nov 1;67(3):483–493. doi: 10.1016/0092-8674(91)90523-2. [DOI] [PubMed] [Google Scholar]
  41. Paz-Ares J., Ghosal D., Wienand U., Peterson P. A., Saedler H. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J. 1987 Dec 1;6(12):3553–3558. doi: 10.1002/j.1460-2075.1987.tb02684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Romero I., Fuertes A., Benito M. J., Malpica J. M., Leyva A., Paz-Ares J. More than 80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant J. 1998 May;14(3):273–284. doi: 10.1046/j.1365-313x.1998.00113.x. [DOI] [PubMed] [Google Scholar]
  43. Rosinski J. A., Atchley W. R. Molecular evolution of the Myb family of transcription factors: evidence for polyphyletic origin. J Mol Evol. 1998 Jan;46(1):74–83. doi: 10.1007/pl00006285. [DOI] [PubMed] [Google Scholar]
  44. Sablowski R. W., Moyano E., Culianez-Macia F. A., Schuch W., Martin C., Bevan M. A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. EMBO J. 1994 Jan 1;13(1):128–137. doi: 10.1002/j.1460-2075.1994.tb06242.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Speulman E., Metz P. L., van Arkel G., te Lintel Hekkert B., Stiekema W. J., Pereira A. A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome. Plant Cell. 1999 Oct;11(10):1853–1866. doi: 10.1105/tpc.11.10.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C. The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco . Plant Cell. 1998 Feb;10(2):135–154. doi: 10.1105/tpc.10.2.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Thompson M. A., Ramsay R. G. Myb: an old oncoprotein with new roles. Bioessays. 1995 Apr;17(4):341–350. doi: 10.1002/bies.950170410. [DOI] [PubMed] [Google Scholar]
  48. Tissier A. F., Marillonnet S., Klimyuk V., Patel K., Torres M. A., Murphy G., Jones J. D. Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics. Plant Cell. 1999 Oct;11(10):1841–1852. doi: 10.1105/tpc.11.10.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Urao T., Yamaguchi-Shinozaki K., Urao S., Shinozaki K. An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell. 1993 Nov;5(11):1529–1539. doi: 10.1105/tpc.5.11.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Waites R., Selvadurai H. R., Oliver I. R., Hudson A. The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum. Cell. 1998 May 29;93(5):779–789. doi: 10.1016/s0092-8674(00)81439-7. [DOI] [PubMed] [Google Scholar]
  51. Winkler R. G., Frank M. R., Galbraith D. W., Feyereisen R., Feldmann K. A. Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in the cytochrome p450 gene superfamily. Plant Physiol. 1998 Nov;118(3):743–750. doi: 10.1104/pp.118.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wisman E., Cardon G. H., Fransz P., Saedler H. The behaviour of the autonomous maize transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis. Plant Mol Biol. 1998 Aug;37(6):989–999. doi: 10.1023/a:1006082009151. [DOI] [PubMed] [Google Scholar]
  53. Wisman E., Hartmann U., Sagasser M., Baumann E., Palme K., Hahlbrock K., Saedler H., Weisshaar B. Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12432–12437. doi: 10.1073/pnas.95.21.12432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yang Y., Klessig D. F. Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14972–14977. doi: 10.1073/pnas.93.25.14972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zwaal R. R., Broeks A., van Meurs J., Groenen J. T., Plasterk R. H. Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7431–7435. doi: 10.1073/pnas.90.16.7431. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES