, Volume 244, Issue 2, pp 467–478 | Cite as

Isolation and characterization of the Jatropha curcas APETALA1 (JcAP1) promoter conferring preferential expression in inflorescence buds

  • Yan-Bin Tao
  • Liang-Liang He
  • Longjian Niu
  • Zeng-Fu XuEmail author
Original Article


Main conclusion

The 1.5 kb JcAP1 promoter from the biofuel plant Jatropha curcas is predominantly active in the inflorescence buds of transgenic plants, in which the −1313/−1057 region is essential for maintaining the activity.

Arabidopsis thaliana APETALA1 (AP1) is a MADS-domain transcription factor gene that functions primarily in flower development. We isolated a homolog of AP1 from Jatropha curcas (designated JcAP1), which was shown to exhibit flower-specific expression in Jatropha. JcAP1 is first expressed in inflorescence buds and continues to be primarily expressed in the sepals. We isolated a 1.5 kb JcAP1 promoter and evaluated its activity in transgenic Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis and Jatropha, the inflorescence buds exhibited notable GUS activity, whereas the sepals did not. Against expectations, the JcAP1 promoter was active in the anthers of Arabidopsis and Jatropha and was highly expressed in Jatropha seeds. An analysis of promoter deletions in transgenic Arabidopsis revealed that deletion of the −1313/−1057 region resulted in loss of JcAP1 promoter activity in the inflorescence buds and increased activity in the anthers. These results suggested that some regulatory sequences in the −1313/−1057 region are essential for maintaining promoter activity in inflorescence buds and can partly suppress activity in the anthers. Based on these findings, we hypothesized that other elements located upstream of the 1.5 kb JcAP1 promoter may be required for flower-specific activation. The JcAP1 promoter characterized in this study can be used to drive transgene expression in both the inflorescence buds and seeds of Jatropha.


Anther APETALA1 Flower Physic nut Promoter Seed 



This work was supported by funding from the National Natural Science Foundation of China (31370595), the CAS 135 program (XTBG-T02), and the Top Science and Technology Talents Scheme of Yunnan Province (2009CI123). The authors gratefully acknowledge the Central Laboratory of the Xishuangbanna Tropical Botanical Garden for providing research facilities.


  1. Azeez A, Miskolczi P, Tylewicz S, Bhalerao RP (2014) A tree ortholog of APETALA1 mediates photoperiodic control of seasonal growth. Curr Biol 24:717–724CrossRefPubMedGoogle Scholar
  2. Berbel A, Navarro C, Ferrandiz C, Canas LA, Madueno F, Beltran JP (2001) Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species. Plant J 25:441–451CrossRefPubMedGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  4. Cai Y, Sun D, Wu G, Peng J (2010) ISSR-based genetic diversity of Jatropha curcas germplasm in China. Biomass Bioenerg 34:1739–1750CrossRefGoogle Scholar
  5. Chen M-S, Wang G-J, Wang R-L, Wang J, Song S-Q, Xu Z-F (2011) Analysis of expressed sequence tags from biodiesel plant Jatropha curcas embryos at different developmental stages. Plant Sci 181:696–700CrossRefPubMedGoogle Scholar
  6. Chen M-S, Pan B-Z, Wang G-J, Ni J, Niu L, Xu Z-F (2014) Analysis of the transcriptional responses in inflorescence buds of Jatropha curcas exposed to cytokinin treatment. BMC Plant Biol 14:318CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chi Y, Huang F, Liu H, Yang S, Yu D (2011) An APETALA1-like gene of soybean regulates flowering time and specifies floral organs. J Plant Physiol 168:2251–2259CrossRefPubMedGoogle Scholar
  8. Chou M-L, Haung M-D, Yang C-H (2001) EMF genes interact with late-flowering genes in regulating floral initiation genes during shoot development in Arabidopsis thaliana. Plant Cell Physiol 42:499–507CrossRefPubMedGoogle Scholar
  9. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  10. Coen ES, Meyerowitz EM (1991) The war of the whorls—genetic interactions controlling flower development. Nature 353:31–37CrossRefPubMedGoogle Scholar
  11. de Argollo Marques D, Siqueira WJ, Colombo CA, Ferrari RA (2013) Breeding and biotechnology of Jatropha curcas. In: Bahadur B, Sujatha M, Carels N (eds) Jatropha, challenges for a new energy crop. Volume 2: genetic improvement and biotechnology. Springer, New York, pp 457–478CrossRefGoogle Scholar
  12. Ding L-W, Sun Q-Y, Wang Z-Y, Sun Y-B, Xu Z-F (2008) Using silica particles to isolate total RNA from plant tissues recalcitrant to extraction in guanidine thiocyanate. Anal Biochem 374:426–428CrossRefPubMedGoogle Scholar
  13. Divakara BN, Upadhyaya HD, Wani SP, Gowda CLL (2010) Biology and genetic improvement of Jatropha curcas L.: a review. Appl Energ 87:732–742CrossRefGoogle Scholar
  14. Douglas CJ, Hauffe KD, Itesmorales ME, Ellard M, Paszkowski U, Hahlbrock K, Dangl JL (1991) Exonic sequences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression. EMBO J 10:1767–1775PubMedPubMedCentralGoogle Scholar
  15. Ellul P, Angosto T, Garcia-Sogo B, Garcia-Hurtado N, Martin-Trillo M, Salinas M, Moreno V, Lozano R, Martinez-Zapater M (2004) Expression of Arabidopsis APETALA1 in tomato reduces its vegetative cycle without affecting plant production. Mol Breed 13:155–163CrossRefGoogle Scholar
  16. Fu Q, Li C, Tang M, Tao Y-B, Pan B-Z, Zhang L, Niu L, He H, Wang X, Xu Z-F (2015) An efficient protocol for Agrobacterium-mediated transformation of the biofuel plant Jatropha curcas by optimizing kanamycin concentration and duration of delayed selection. Plant Biotechnol Rep 9:405–416CrossRefPubMedPubMedCentralGoogle Scholar
  17. Garbarino JE, Belknap WR (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol Biol 24:119–127CrossRefPubMedGoogle Scholar
  18. Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, Barbas CF 3rd, Briggs SP (2002) Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc Natl Acad Sci USA 99:13296–13301CrossRefPubMedPubMedCentralGoogle Scholar
  19. Heller J (1996) Physic nut Jatropha curcas L. Promoting the conservation and use of underutilized and neglected crops. 1. Institute of Plant Genetics and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute, RomeGoogle Scholar
  20. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300CrossRefPubMedPubMedCentralGoogle Scholar
  21. Huang H, Wang S, Jiang J, Liu G, Li H, Chen S, Xu H (2014) Overexpression of BpAP1 induces early flowering and produces dwarfism in Betula platyphylla x Betula pendula. Physiol Plant 151:495–506CrossRefPubMedGoogle Scholar
  22. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in plants. EMBO J 6:3901–3907PubMedPubMedCentralGoogle Scholar
  23. Jeong YM, Jung EJ, Hwang HJ, Kim H, Lee SY, Kim SG (2009) Roles of the first intron on the expression of Arabidopsis (Arabidopsis thaliana) genes for actin and actin-binding proteins. Plant Sci 176:58–65CrossRefGoogle Scholar
  24. Joshi M, Mishra A, Jha B (2010) Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes. Ind Crops Prod 33:67–77CrossRefGoogle Scholar
  25. Kaufmann K, Wellmer F, Muino JM, Ferrier T, Wuest SE, Kumar V, Serrano-Mislata A, Madueno F, Krajewski P, Meyerowitz EM, Angenent GC, Riechmann JL (2010) Orchestration of floral initiation by APETALA1. Science 328:85–89CrossRefPubMedGoogle Scholar
  26. Kawagoe Y, Murai N (1992) Four distinct nuclear proteins recognize in vitro the proximal promoter of the bean seed storage protein β-phaseolin gene conferring spatial and temporal control. Plant J 2:927–936PubMedGoogle Scholar
  27. Kim MJ, Yang SW, Mao H-Z, Veena SP, Yin J-L, Chua N-H (2014) Gene silencing of Sugar-dependent 1 (JcSDP1), encoding a patatin-domain triacylglycerol lipase, enhances seed oil accumulation in Jatropha curcas. Biotechnol Biofuels 7:36CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kotoda N, Wada M, Kusaba S, Kano-Murakami Y, Masuda T, Soejima J (2002) Overexpression of MdMADS5, an APETALA1-like gene of apple, causes early flowering in transgenic Arabidopsis. Plant Sci 162:679–687CrossRefGoogle Scholar
  29. Kramer EM, Dorit RL, Irish VF (1998) Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages. Genetics 149:765–783PubMedPubMedCentralGoogle Scholar
  30. Kumar N, Anand KGV, Pamidimarri D, Sarkar T, Reddy MP, Radhakrishnan T, Kaul T, Reddy MK, Sopori SK (2010) Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants. Ind Crop Prod 32:41–47CrossRefGoogle Scholar
  31. Lessard PA, Allen RD, Bernier F, Crispino JD, Fujiwara T, Beachy RN (1991) Multiple nuclear factors interact with upstream sequences of differentially regulated β-conglycinin genes. Plant Mol Biol 16:397–413CrossRefPubMedGoogle Scholar
  32. Li M, Li H, Jiang H, Pan X, Wu G (2008) Establishment of an Agrobacteriuim-mediated cotyledon disc transformation method for Jatropha curcas. Plant Cell Tiss Org 92:173–181CrossRefGoogle Scholar
  33. Li XG, Su YH, Zhao XY, Li W, Gao XQ, Zhang XS (2010) Cytokinin overproduction-caused alteration of flower development is partially mediated by CUC2 and CUC3 in Arabidopsis. Gene 450:109–120CrossRefPubMedGoogle Scholar
  34. Li C, Luo L, Fu Q, Niu L, Xu Z-F (2014) Isolation and functional characterization of JcFT, a FLOWERING LOCUS T (FT) homologous gene from the biofuel plant Jatropha curcas. BMC Plant Biol 14:125CrossRefPubMedPubMedCentralGoogle Scholar
  35. Li CQ, Luo L, Fu QT, Niu LJ, Xu ZF (2015) Identification and characterization of the FT/TFL1 gene family in the biofuel plant Jatropha curcas. Plant Mol Biol Rep 33:326–333CrossRefGoogle Scholar
  36. Litt A, Kramer EM (2010) The ABC model and the diversification of floral organ identity. Semin Cell Dev Biol 21:129–137CrossRefPubMedGoogle Scholar
  37. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463CrossRefPubMedGoogle Scholar
  38. Mandel MA, Gustafsonbrown C, Savidge B, Yanofsky MF (1992) Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature 360:273–277CrossRefPubMedGoogle Scholar
  39. Mena M, Mandel MA, Lerner DR, Yanofsky MF, Schmidt RJ (1995) A characterization of the MADS-box gene family in maize. Plant J 8:845–854CrossRefPubMedGoogle Scholar
  40. Murai K, Miyamae M, Kato H, Takumi S, Ogihara Y (2003) WAP1, a wheat APETALA1 homolog, plays a central role in the phase transition from vegetative to reproductive growth. Plant Cell Physiol 44:1255–1265CrossRefPubMedGoogle Scholar
  41. Muschietti J, Dircks L, Vancanneyt G, Mccormick S (1994) Lat52 protein is essential for tomato pollen development: pollen expressing antisense Lat52 RNA hydrates and germinates abnormally and cannot achieve fertilization. Plant J 6:321–338CrossRefPubMedGoogle Scholar
  42. Pan BZ, Xu ZF (2011) Benzyladenine treatment significantly increases the seed yield of the biofuel plant Jatropha curcas. J Plant Growth Regul 30:166–174CrossRefGoogle Scholar
  43. Pan J, Fu Q, Xu ZF (2010) Agrobacterium tumefaciens-mediated transformation of biofuel plant Jatropha curcas using kanamycin selection. Afr J Biotechnol 9:6477–6481Google Scholar
  44. Pan B-Z, Chen M-S, Ni J, Xu Z-F (2014) Transcriptome of the inflorescence meristems of the biofuel plant Jatropha curcas treated with cytokinin. BMC Genom 15:974CrossRefGoogle Scholar
  45. Pena L, Martin-Trillo M, Juarez J, Pina JA, Navarro L, Martinez-Zapater JM (2001) Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nat Biotechnol 19:263–267CrossRefPubMedGoogle Scholar
  46. Pillitteri LJ, Lovatt CJ, Walling LL (2004) Isolation and characterization of LEAFY and APETALA1 homologues from Citrus sinensis L. Osbeck ‘Washington’. J Am Soc Hortic Sci 129:846–856Google Scholar
  47. Qin XB, Zhang JP, Shao CX, Lin S, Jiang LD, Zhang SW, Xu Y, Chen F (2009a) Isolation and characterization of a curcin promoter from Jatropha curcas L. and its regulation of gene expression in transgenic tobacco plants. Plant Mol Biol Rep 27:275–281CrossRefGoogle Scholar
  48. Qin XB, Zheng XJ, Shao CX, Gao JH, Jiang LD, Zhu XL, Yan F, Tang L, Xu Y, Chen F (2009b) Stress-induced curcin-L promoter in leaves of Jatropha curcas L. and characterization in transgenic tobacco. Planta 230:387–395CrossRefPubMedGoogle Scholar
  49. Qin XB, Zheng XJ, Huang XQ, Lii YF, Shao CX, Xu Y, Chen F (2014) A novel transcription factor JcNAC1 response to stress in new model woody plant Jatropha curcas. Planta 239:511–520CrossRefPubMedGoogle Scholar
  50. Qu J, Mao HZ, Chen W, Gao SQ, Bai YN, Sun YW, Geng YF, Ye J (2012) Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid. Biotechnol Biofuels 5:10CrossRefPubMedPubMedCentralGoogle Scholar
  51. Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale DM, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585CrossRefPubMedGoogle Scholar
  52. Sasaki K, Yamaguchi H, Narumi T, Shikata M, Oshima Y, Nakata M, Mitsuda N, Ohme-Takagi M, Ohtsubo N (2011) Utilization of a floral organ-expressing AP1 promoter for generation of new floral traits in Torenia fournieri Lind. Plant Biotechnol 28:181–188CrossRefGoogle Scholar
  53. Sato S, Hirakawa H, Isobe S, Fukai E, Watanabe A, Kato M, Kawashima K, Minami C, Muraki A, Nakazaki N, Takahashi C, Nakayama S, Kishida Y, Kohara M, Yamada M, Tsuruoka H, Sasamoto S, Tabata S, Aizu T, Toyoda A, Shin-i T, Minakuchi Y, Kohara Y, Fujiyama A, Tsuchimoto S, Kajiyama S, Makigano E, Ohmido N, Shibagaki N, Cartagena JA, Wada N, Kohinata T, Atefeh A, Yuasa S, Matsunaga S, Fukui K (2011) Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L. DNA Res 18:65–76CrossRefPubMedGoogle Scholar
  54. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088CrossRefPubMedPubMedCentralGoogle Scholar
  55. Stålberg K, Ellerstöm M, Ezcurra I, Ablov S, Rask L (1996) Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta 199:515–519CrossRefPubMedGoogle Scholar
  56. Sujatha M, Reddy TP, Mahasi MJ (2008) Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L. Biotechnol Adv 26:424–435CrossRefPubMedGoogle Scholar
  57. Tang MJ, Liu XF, Deng HP, Shen SH (2011) Over-expression of JcDREB, a putative AP2/EREBP domain-containing transcription factor gene in woody biodiesel plant Jatropha curcas, enhances salt and freezing tolerance in transgenic Arabidopsis thaliana. Plant Sci 181:623–631CrossRefPubMedGoogle Scholar
  58. Tao YB, Luo L, He LL, Ni J, Xu ZF (2014) A promoter analysis of MOTHER OF FT AND TFL1 1 (JcMFT1), a seed-preferential gene from the biofuel plant Jatropha curcas. J Plant Res 127:513–524CrossRefPubMedGoogle Scholar
  59. Tao YB, He LL, Niu LJ, Xu ZF (2015) Isolation and characterization of an ubiquitin extension protein gene (JcUEP) promoter from Jatropha curcas. Planta 241:823–836CrossRefPubMedGoogle Scholar
  60. Tatikonda L, Wani SP, Kannan S, Beerelli N, Sreedevi TK, Hoisington DA, Devi P, Varshney RK (2009) AFLP-based molecular characterization of an elite germplasm collection of Jatropha curcas L., a biofuel plant. Plant Sci 176:505–513CrossRefPubMedGoogle Scholar
  61. Tilly JJ, Allen DW, Jack T (1998) The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects. Development 125:1647–1657PubMedGoogle Scholar
  62. Twell D, Yamaguchi J, Wing RA, Ushiba J, Mccormick S (1991) Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Gene Dev 5:496–507CrossRefPubMedGoogle Scholar
  63. Wagner D, Sablowski RWM, Meyerowitz EM (1999) Transcriptional activation of APETALA1 by LEAFY. Science 285:582–584CrossRefPubMedGoogle Scholar
  64. Wei Q, Li J, Zhang L, Wu P, Chen Y, Li M, Jiang H, Wu G (2012) Cloning and characterization of a β-ketoacyl-acyl carrier protein synthase II from Jatropha curcas. J Plant Physiol 169:816–824CrossRefPubMedGoogle Scholar
  65. Yanagisawa S, Schmidt RJ (1999) Diversity and similarity among recognition sequences of Dof transcription factors. Plant J 17:209–214CrossRefPubMedGoogle Scholar
  66. Yun JY, Weigel D, Lee I (2002) Ectopic expression of SUPERMAN suppresses development of petals and stamens. Plant Cell Physiol 43:52–57CrossRefPubMedGoogle Scholar
  67. Zhang L, He L-L, Fu Q-T, Xu Z-F (2013) Selection of reliable reference genes for gene expression studies in the biofuel plant Jatropha curcas using real-time quantitative PCR. Int J Mol Sci 14:24338–24354CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Yan-Bin Tao
    • 1
  • Liang-Liang He
    • 1
    • 2
  • Longjian Niu
    • 1
    • 3
  • Zeng-Fu Xu
    • 1
    Email author
  1. 1.Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical GardenChinese Academy of SciencesMenglaChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.School of Life SciencesUniversity of Science and Technology of ChinaHefeiChina

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