The BRANCHING ENZYME1 gene, encoding a glycoside hydrolase family 13 protein, is required for in vitro plant regeneration in Arabidopsis

  • Xingchun WangEmail author
  • Zhirong Yang
  • Min Wang
  • Lingzhi Meng
  • Yiwei Jiang
  • Yuanhuai HanEmail author
Original Paper


In vitro plant regeneration requires the coordinated action of various enzymes in addition to phytohormones. Here, we report that the Arabidopsis Branching Enzyme 1 (BE1) gene, encoding a putative glycoside hydrolase involved in carbohydrate metabolism, is critical for explant regeneration. A partial loss-of-function mutation of the BE1 gene (be1-3 mutant) severely impaired adventitious shoot formation and somatic embryogenesis but not root formation in tissue culture. An in planta hormone response assay revealed that be1-3 seedlings showed normal response to cytokinin and auxin. The calli formed from be1-3 mutants were less plump than those of wild type hypocotyls. The BE1 gene is mainly expressed in the xylem pericycle of the hypocotyl and root and in dedifferentiated and differentiating calli. Expression levels of BE1 decreases gradually during shoot formation. Consistent with its role in carbohydrate metabolism, mutation of the BE1 gene dramatically reduces the content of glucose and fructose in seeds. Transcriptomic profiles showed 1,860 and 832 differentially expressed genes between the mutant and wild type during callus and shoot development, respectively. Most of them were related to metabolism, hormone signal transduction and stress response. These results indicate that the BE1 gene is involved in organogenesis and somatic embryogenesis by regulating carbohydrate metabolism.


Arabidopsis thaliana Explant regeneration Shoot organogenesis Somatic embryogenesis Carbohydrate metabolism 



This work was partially done while Xingchun Wang was at Dr. Jianru Zuo’s lab of Institute of Genetics and Developmental Biology. We wish to thank Dr. Zuo and his lab members for their constructive suggestions and technical assistance. We are grateful to Dr. Chunlai Li of the Institute of Genetics and Developmental Biology for help with sugar content determination. We also thank Craig Schluttenhofer of the University of Kentucky for critical reading of the manuscript. This study was supported by the National Natural Science Foundation of China (31100235 and 31171181), Natural Science Foundation of Shanxi (2013011028-1 and 2010021030-1) and China Postdoctoral Science Foundation (80839).

Supplementary material

11240_2014_439_MOESM1_ESM.xls (1 mb)
Supplementary Table 1 Differentially expressed genes during dedifferentiation. (XLS 1,039 kb)
11240_2014_439_MOESM2_ESM.xls (489 kb)
Supplementary Table 2 Differentially expressed genes during redifferentiation. (XLS 489 kb)
11240_2014_439_MOESM3_ESM.tif (6.7 mb)
Supplementary Fig. 1 BE1 is not a target of WUS. a RT-PCR analysis of the expression of BE1 in 2-week-old wus-1 and wild type (WT) seedlings. b RT-PCR analysis of the expression of BE1 in 2-week-old pga6 seedling treated with 10 μM 17-β-estradiol for 0, 12, 24, 48 and 72 h. c Two-week-old seedlings germinated and grown on MS medium in the presence or the absence of 10 μM 17-β-estradiol. From left to right: pga6 on MS medium, pga6 be1-3 on MS medium, pga6 germinated on estradiol-containing medium, and pga6 be1-3 germinated on estradiol-containing medium. Scale bars: 5 mm. (TIFF 6,896 kb)


  1. Abel S, Theologis A (1996) Early genes and auxin action. Plant Physiol 111:9–17PubMedCentralPubMedCrossRefGoogle Scholar
  2. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25:25–29PubMedCentralPubMedCrossRefGoogle Scholar
  3. Atta R, Laurens L, Boucheron-Dubuisson E, Guivarc’h A, Carnero E, Giraudat-Pautot V, Rech P, Chriqui D (2009) Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. Plant J 57:626–644PubMedCrossRefGoogle Scholar
  4. Berleth T, Krogan NT, Scarpella E (2004) Auxin signals-turning genes on and turning cells around. Curr Opin Plant Biol 7:553–563PubMedCrossRefGoogle Scholar
  5. Che P, Gingerich DJ, Lall S, Howell SH (2002) Global and hormone-induced gene expression changes during shoot development in Arabidopsis. Plant Cell 14:2771–2785PubMedCentralPubMedCrossRefGoogle Scholar
  6. Che P, Lall S, Nettleton D, Howell SH (2006) Gene expression programs during shoot, root, and callus development in Arabidopsis tissue culture. Plant Physiol 141:620–637PubMedCentralPubMedCrossRefGoogle Scholar
  7. Christianson ML, Warnick DA (1983) Competence and determination in the process of in vitro shoot organogenesis. Dev Biol 95:288–293PubMedCrossRefGoogle Scholar
  8. Daimon Y, Takabe K, Tasaka M (2003) The CUP-SHAPED COTYLEDON genes promote adventitious shoot formation on calli. Plant Cell Physiol 44:113–121PubMedCrossRefGoogle Scholar
  9. Estelle MA, Somerville C (1987) Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol Gen Genet 206:200–206CrossRefGoogle Scholar
  10. Fan M, Xu C, Xu K, Hu Y (2012) LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration. Cell Res 22:1169–1180PubMedCentralPubMedCrossRefGoogle Scholar
  11. Gaj MD (2001) Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana. Plant Cell, Tissue Organ Cult 64:39–46CrossRefGoogle Scholar
  12. Gaj MD (2011) Somatic embryogenesis and plant regeneration in the culture of Arabidopsis thaliana (L.) Heynh. immature zygotic embryos. In: Thorpe TA, Yeung EC (eds) Plant embryo culture: methods and protocols. Humana Press, New York, pp 257–265CrossRefGoogle Scholar
  13. Gaj MD, Zhang S, Harada JJ, Lemaux PG (2005) Leafy cotyledon genes are essential for induction of somatic embryogenesis of Arabidopsis. Planta 222:977–988PubMedCrossRefGoogle Scholar
  14. Gallois JL, Nora FR, Mizukami Y, Sablowski R (2004) WUSCHEL induces shoot stem cell activity and developmental plasticity in the root meristem. Gene Dev 18:375–380PubMedCentralPubMedCrossRefGoogle Scholar
  15. Gliwicka M, Nowak K, Balazadeh S, Mueller-Roeber B, Gaj MD (2013) Extensive modulation of the transcription factor transcriptome during somatic embryogenesis in Arabidopsis thaliana. PLoS ONE 8:e69261PubMedCentralPubMedCrossRefGoogle Scholar
  16. Gordon SP, Heisler MG, Reddy GV, Ohno C, Das P, Meyerowitz EM (2007) Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development 134:3539–3548PubMedCrossRefGoogle Scholar
  17. Higuchi M, Pischke MS, Mahonen AP, Miyawaki K, Hashimoto Y, Seki M, Kobayashi M, Shinozaki K, Kato T, Tabata S, Helariutta Y, Sussman MR, Kakimoto T (2004) In planta functions of the Arabidopsis cytokinin receptor family. Proc Natl Acad Sci USA 101:8821–8826PubMedCentralPubMedCrossRefGoogle Scholar
  18. Ikeda Y, Banno H, Niu QW, Howell SH, Chua NH (2006) The ENHANCER OF SHOOT REGENERATION 2 gene in Arabidopsis regulates CUP-SHAPED COTYLEDON 1 at the transcriptional level and controls cotyledon development. Plant Cell Physiol 47:1443–1456PubMedCrossRefGoogle Scholar
  19. Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata S, Shinozaki K, Kakimoto T (2001) Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409:1060–1063PubMedCrossRefGoogle Scholar
  20. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedCentralPubMedGoogle Scholar
  21. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:D480–D484PubMedCentralPubMedCrossRefGoogle Scholar
  22. Long JA, Moan EI, Medford JI, Barton MK (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379:66–69PubMedCrossRefGoogle Scholar
  23. Matsuo N, Makino M, Banno H (2011) Arabidopsis ENHANCER OF SHOOT REGENERATION (ESR)1 and ESR2 regulate in vitro shoot regeneration and their expressions are differentially regulated. Plant Sci 181:39–46PubMedCrossRefGoogle Scholar
  24. Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T (1998) Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95:805–815PubMedCrossRefGoogle Scholar
  25. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628PubMedCrossRefGoogle Scholar
  26. Motte H, Galuszka P, Spichal L, Tarkowski P, Plihal O, Smehilova M, Jaworek P, Vereecke D, Werbrouck S, Geelen D (2013) Phenyl-adenine, identified in a LIGHT-DEPENDENT SHORT HYPOCOTYLS4-assisted chemical screen, is a potent compound for shoot regeneration through the inhibition of CYTOKININ OXIDASE/DEHYDROGENASE activity. Plant Physiol 161:1229–1241PubMedCentralPubMedCrossRefGoogle Scholar
  27. Ogawa D, Yamaguchi K, Nishiuchi T (2007) High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth. J Exp Bot 58:3373–3383PubMedCrossRefGoogle Scholar
  28. Sakai H, Honma T, Aoyama T, Sato S, Kato T, Tabata S, Oka A (2001) ARR1, a transcription factor for genes immediately responsive to cytokinins. Science 294:1519–1521PubMedCrossRefGoogle Scholar
  29. Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–130PubMedGoogle Scholar
  30. Sugimoto K, Jiao Y, Meyerowitz EM (2010) Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell 18:463–471PubMedCrossRefGoogle Scholar
  31. Sulpice R, Pyl E-T, Ishihara H, Trenkamp S, Steinfath M, Witucka-Wall H, Gibon Y, Usadel B, Poree F, Piques MC, Von Korff M, Steinhauser MC, Keurentjes JJB, Guenther M, Hoehne M, Selbig J, Fernie AR, Altmann T, Stitt M (2009) Starch as a major integrator in the regulation of plant growth. Proc Natl Acad Sci 106:10348–10353PubMedCentralPubMedCrossRefGoogle Scholar
  32. Sun J, Niu QW, Tarkowski P, Zheng B, Tarkowska D, Sandberg G, Chua NH, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol 131:167–176PubMedCentralPubMedCrossRefGoogle Scholar
  33. Sun J, Hirose N, Wang X, Wen P, Xue L, Sakakibara H, Zuo J (2005) Arabidopsis SOI33/AtENT8 gene encodes a putative equilibrative nucleoside transporter that is involved in cytokinin transport in planta. J Integr Plant Biol 47:588–603CrossRefGoogle Scholar
  34. Valvekens D, Montagu MV, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540PubMedCentralPubMedCrossRefGoogle Scholar
  35. Veyres N, Danon A, Aono M, Galliot S, Karibasappa YB, Diet A, Grandmottet F, Tamaoki M, Lesur D, Pilard S, Boitel-Conti M, Sangwan-Norreel BS, Sangwan RS (2008) The Arabidopsis sweetie mutant is affected in carbohydrate metabolism and defective in the control of growth, development and senescence. Plant J 55:665–686PubMedCrossRefGoogle Scholar
  36. Wang X, Xue L, Sun J, Zuo J (2010) The Arabidopsis BE1 gene, encoding a putative glycoside hydrolase localized in plastids, plays crucial roles during embryogenesis and carbohydrate metabolism. J Integr Plant Biol 52:273–288PubMedCrossRefGoogle Scholar
  37. Yasutani I, Ozawa S, Nishida T, Sugiyama M, Komamine A (1994) Isolation of temperature-sensitive mutants of Arabidopsis thaliana that are defective in the redifferentiation of shoots. Plant Physiol 105:815–822PubMedCentralPubMedGoogle Scholar
  38. Zhao X, Su Y, Zhang C, Wang L, Li X, Zhang X (2013) Differences in capacities of in vitro organ regeneration between two Arabidopsis ecotypes Wassilewskija and Columbia. Plant Cell, Tissue Organ Cult 112:65–74CrossRefGoogle Scholar
  39. Zuo J, Niu QW, Frugis G, Chua NH (2002) The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis. Plant J 30:349–359PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.College of Life SciencesShanxi Agricultural UniversityTaiguChina
  2. 2.Institute of Agricultural BioengineeringShanxi Agricultural UniversityTaiguChina
  3. 3.Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess PlateauMinistry of AgricultureTaiyuanChina
  4. 4.Department of AgronomyPurdue UniversityWest LafayetteUSA

Personalised recommendations