Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits

  • Yi-Hong Wang
  • Chris M. Mosebach
  • Abraham S. Kibbey
  • Marcie K. Ryhal
  • Angelica D. Jones
  • Julie A. Palmer
  • Leon V. Kochian


Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.


Full-length cDNA library Binary vector Plant transformation Tomato Arabidopsis thaliana Heterologous expression 



The project was supported by Penn State Erie, The Behrend College. CMM, ASK, MKR, and ADJ were supported in part by Penn State Behrend Undergraduate Student Academic Year Research Grant Program and Undergraduate Student Summer Research Fellowship Program. Daryl J. Nowacki participated in the early phase of the project. We thank Michael A. Campbell and James T. Warren for reviewing the manuscript.


  1. Bucher M, Schroeer B, Willmitzer L, Riesmeier JW (1997) Two genes encoding extension-like proteins are predominantly expressed in tomato root hair cells. Plant Mol Biol 35:497–508. doi: 10.1023/A:1005869717158 CrossRefPubMedGoogle Scholar
  2. Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287–293. doi: 10.1023/A:1023475710642 CrossRefGoogle Scholar
  3. Choe S, Tanaka A, Noguchi T, Fujioka S, Takatsuto S, Ross AS, Tax FE, Yoshida S, Feldmann KA (2000) Lesions in the sterol Δ7 reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis. Plant J 21:431–443. doi: 10.1046/j.1365-313x.2000.00693.x CrossRefPubMedGoogle Scholar
  4. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743. doi: 10.1046/j.1365-313x.1998.00343.x CrossRefPubMedGoogle Scholar
  5. Diener A, Hirschi K (2000) Heterologous expression for dominant-like gene activity. Trends Plant Sci 5:10–11. doi: 10.1016/S1360-1385(99) 01512-5 CrossRefPubMedGoogle Scholar
  6. Feldmann KA (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J 1:71–82. doi: 10.1111/j.1365-313X.1991.00071.x CrossRefGoogle Scholar
  7. Fujita M, Mizukado S, Fujita Y, Ichikawa T, Nakazawa M, Seki M, Matsui M, Yamaguchi-Shinozaki K, Shinozaki K (2007) Identification of stress-tolerance-related transcription-factor genes via mini-scale Full-length cDNA Over-eXpressor (FOX) gene hunting system. Biochem Biophys Res Commun 364:250–257. doi: 10.1016/j.bbrc.2007.09.124 CrossRefPubMedGoogle Scholar
  8. Gillaspy G, Ben-David H, Gruissem W (1993) Fruits: a developmental perspective. Plant Cell 5:1439–1451CrossRefPubMedGoogle Scholar
  9. González-Carranza ZH, Rompa U, Peters JL, Bhatt AM, Wagstaff C, Stead AD, Roberts JA (2007) Hawaiian skirt: an F-box gene that regulates organ fusion and growth in Arabidopsis. Plant Physiol 144:1370–1382. doi: 10.1104/pp. 106.092288 CrossRefPubMedGoogle Scholar
  10. Greenboim-Wainberg Y, Maymon I, Borochov R, Alvarez J, Olszewski N, Ori N, Eshed Y, Weiss D (2005) Cross talk between Gibberellin and Cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling. Plant Cell 17:92–102. doi: 10.1105/tpc.104.028472 CrossRefPubMedGoogle Scholar
  11. Hanson AD, Gage DA, Shachar-Hill Y (2000) Plant one-carbon metabolism and its engineering. Trends Plant Sci 5:206–213. doi: 10.1016/S1360-1385(00)01599-5 CrossRefPubMedGoogle Scholar
  12. Helliwell CA, Sheldon CC, Olive MR, Walker AR, Zeevaart JA, Peacock WJ, Dennis ES (1998) Cloning of the Arabidopsis ent-kaurene oxidase gene GA3. Proc Natl Acad Sci U S A 95:9019–9024. doi: 10.1073/pnas.95.15.9019 CrossRefPubMedGoogle Scholar
  13. Hoffman PD, Leonard JM, Lindberg GE, Bollmann SR, Hays JB (2004) Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis. Genes Dev 18:2676–2685. doi: 10.1101/gad.1217204 CrossRefPubMedGoogle Scholar
  14. Ichikawa T, Nakazawa M, Kawashima M, Iizumi H, Kuroda H, Kondou Y, Tsuhara Y, Suzuki K, Ishikawa A, Seki M, Fujita M, Motohashi R, Nagata N, Takagi T, Shinozaki K, Matsui M (2006) The FOX hunting system: an alternative gain-of-function gene hunting technique. Plant J 48:974–985. doi: 10.1111/j.1365-313X.2006.02924.x CrossRefPubMedGoogle Scholar
  15. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  16. Jenks MA, Rashotte AM, Tuttle HA, Feldmann KA (1996) Mutants in Arabidopsis thaliana altered in epicuticular wax and leaf morphology. Plant Physiol 110:377–385PubMedGoogle Scholar
  17. Lai MD, Xu J (2007) Ribosomal proteins and colorectal cancer. Curr Genomics 8:43–49. doi: 10.2174/138920207780076938 CrossRefPubMedGoogle Scholar
  18. LeClere S, Bartel B (2001) A library of Arabidopsis 35S-cDNA lines for identifying novel mutants. Plant Mol Biol 46:695–703. doi: 10.1023/A:1011699722052 CrossRefPubMedGoogle Scholar
  19. Lein W, Usadel B, Stitt M, Reindl A, Ehrhardt T, Sonnewald U, Börnke F (2008) Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions. Plant Biotechnol J 6:246–263. doi: 10.1111/j.1467-7652.2007.00313.x CrossRefPubMedGoogle Scholar
  20. Lu C, Wallis JG, Browse J (2007) An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library. BMC Plant Biol 7:42. doi: 10.1186/1471-2229-7-42 CrossRefPubMedGoogle Scholar
  21. Nakamura H, Hakata M, Amano K, Miyao A, Toki N, Kajikawa M, Pang J, Higashi N, Ando S, Toki S, Fujita M, Enju A, Seki M, Nakazawa M, Ichikawa T, Shinozaki K, Matsui M, Nagamura Y, Hirochika H, Ichikawa H (2007) A genome-wide gain-of function analysis of rice genes using the FOX-hunting system. Plant Mol Biol 65:357–3571. doi: 10.1007/s11103-007-9243-y CrossRefPubMedGoogle Scholar
  22. Obiadalla-Ali H, Fernie AR, Kossmann J, Lloyd JR (2004) Developmental analysis of carbohydrate metabolism in tomato (Lycopersicon esculentum cv. Micro-Tom) fruits. Physiol Plant 120:196–204. doi: 10.1111/j.0031-9317.2004.0167.x CrossRefPubMedGoogle Scholar
  23. Pinon V, Etchells JP, Rossignol P, Collier SA, Arroyo JM, Martienssen RA, Byrne ME (2008) Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135:1315–1324. doi: 10.1242/dev.016469 CrossRefPubMedGoogle Scholar
  24. Prigge MJ, Wagner DR (2001) The Arabidopsis SERRATE gene encodes a zinc-finger protein required for normal shoot development. Plant Cell 13:1263–1280CrossRefPubMedGoogle Scholar
  25. Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, Sakurai T, Nakajima M, Enju A, Akiyama K, Oono Y, Muramatsu M, Hayashizaki Y, Kawai J, Carninci P, Itoh M, Ishii Y, Arakawa T, Shibata K, Shinagawa A, Shinozaki K (2002) Functional annotation of a full-length Arabidopsis cDNA collection. Science 296:141–145. doi: 10.1126/science.1071006 CrossRefPubMedGoogle Scholar
  26. Schmitz RJ, Sung S, Amasino RM (2008) Histone arginine methylation is required for vernalization-induced epigenetic silencing of FLC in winter-annual Arabidopsis thaliana. Proc Natl Acad Sci U S A 105:411–416. doi: 10.1073/pnas.0710423104 CrossRefPubMedGoogle Scholar
  27. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088. doi: 10.1093/nar/23.6.1087 CrossRefPubMedGoogle Scholar
  28. Stacey NJ, Kuromori T, Azumi Y, Roberts G, Breuer C, Wada T, Maxwell A, Roberts K, Sugimoto-Shirasu K (2006) Arabidopsis SPO11–2 functions with SPO11–1 in meiotic recombination. Plant J 48:206–216. doi: 10.1111/j.1365-313X.2006.02867.x CrossRefPubMedGoogle Scholar
  29. Tian L, Chen ZJ (2001) Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development. Proc Natl Acad Sci U S A 98:200–205. doi: 10.1073/pnas.011347998 CrossRefPubMedGoogle Scholar
  30. Vivian-Smith A, Luo M, Chaudhury A, Koltunow A (2001) Fruit development is actively restricted in the absence of fertilization in Arabidopsis. Development 128:2321–2331PubMedGoogle Scholar
  31. Wang YH, Garvin DF, Kochian LV (2001) Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition. Plant Physiol 127:345–359. doi: 10.1104/pp.127.1.345 CrossRefPubMedGoogle Scholar
  32. Wellenreuther R, Schupp I, Poustka A, Wiemann S (2004) The German cDNA Consortium. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics 5:36. doi: 10.1186/1471-2164-5-36 CrossRefPubMedGoogle Scholar
  33. Yamamoto N, Tsugane T, Watanabe M, Yano K, Maeda F, Kuwata C, Torki M, Ban Y, Nishimura S, Shibata D (2005) Expressed sequence tags from the laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars. Gene 356:127–134. doi: 10.1016/j.gene.2005.04.026 CrossRefPubMedGoogle Scholar
  34. Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30:892–897PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Yi-Hong Wang
    • 1
  • Chris M. Mosebach
    • 1
  • Abraham S. Kibbey
    • 1
  • Marcie K. Ryhal
    • 1
  • Angelica D. Jones
    • 1
  • Julie A. Palmer
    • 1
  • Leon V. Kochian
    • 2
  1. 1.School of SciencePenn State University, Behrend CollegeErieUSA
  2. 2.US Plant Soil and Nutrition LabCornell UniversityIthacaUSA

Personalised recommendations