, Volume 33, Issue 6, pp 1723–1732 | Cite as

A GH3-like gene, LaGH3, isolated from hybrid larch (Larix leptolepis × Larix olgensis) is regulated by auxin and abscisic acid during somatic embryogenesis

  • Li-feng ZhangEmail author
  • Qian Lan
  • Su-ying Han
  • Li-wang QiEmail author
Short Communication
Part of the following topical collections:
  1. Seed Biology and Micropropagation


Key message

A Gretchen Hagen 3-like gene, LaGH3 from hybrid larch (Larix leptolepis × Larix olgensis), was identified, and the effects of abscisic acid (ABA) and indole-3-acetic acid (IAA) on its transcriptional levels and expression patterns during somatic embryogenesis were analyzed.


A full-length cDNA sequence of a Gretchen Hagen 3 (GH3) gene was isolated from hybrid larch (Larix leptolepis × Larix olgensis) and designated LaGH3. The cDNA was 2053 bp in length and contained an 1848-bp open-reading frame encoding a predicted protein of 615 amino acids, characterized by four conserved motifs of the GH3 protein family: P-loop, α5, α6, and β8–β9. The 5′-flanking promoter region of LaGH3 was cloned using an improved TAIL-PCR technique. In this region, we identified several auxin- and abscisic acid (ABA)-inducible elements. Expression analysis showed that LaGH3 was induced by indole-3-acetic acid and that its transcript accumulation was inhibited by ABA. Further experiments demonstrated that ABA was the main factor in LaGH3 downregulation during the early stage of somatic embryogenesis, although the absence of auxin decreased LaGH3 expression slightly. LaGH3 transcripts gradually increased and peaked at 28 days with the rapid development of somatic embryos and then declined as the somatic embryos matured. We suggest that the level of LaGH3 transcripts partly represents the dynamics of endogenous auxin during somatic embryo development in larch. Taken together, our results provide evidence that GH3 genes play important roles in the crosstalk between auxin and ABA by maintaining auxin homeostasis during somatic embryo development.


Conifer Gymnosperm Gretchen Hagen 3 Somatic embryogenesis IAA ABA 



This work was supported by the National Natural Science Foundation of China (31600544 and 31330017), the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2019SY011), and the National Transgenic Major Program of China (2018ZX08020-003).

Author contributions

LZ and LQ designed the experiments and drafted the manuscript; LZ and QL carried out all the experiments and data analysis; SH and LQ proposed and supervised the overall project. All authors read and approved the final version of this manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

468_2019_1904_MOESM1_ESM.doc (5.1 mb)
Supplementary material 1 (DOC 5190 kb)


  1. Bierfreund NM, Tintelnot S, Reski R, Decker EL (2004) Loss of GH3 function does not affect phytochrome-mediated development in a moss, Physcomitrella patens. J Plant Physiol 161:823–835CrossRefPubMedGoogle Scholar
  2. Böttcher C, Keyzers RA, Boss PK, Davies C (2010) Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening. J Exp Bot 61:3615–3625CrossRefPubMedGoogle Scholar
  3. Casanova-Sáez R, Voß U (2019) Auxin metabolism controls developmental decisions in land plants. Trends Plant Sci 24:741–754CrossRefPubMedGoogle Scholar
  4. Chen Q, Westfall CS, Hicks LM, Wang S, Jez JM (2010) Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase. J Biol Chem 285:29780–29786CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chen Q, Zhang B, Hicks LM, Wang S, Jez JM (2009) A liquid chromatography–tandem mass spectrometry-based assay for indole-3-acetic acid–amido synthetase. Anal Biochem 390:149–154CrossRefPubMedGoogle Scholar
  6. Devoghalaere F, Doucen T, Guitton B, Keeling J, Payne W, Ling TJ, Ross JJ, Hallett IC, Gunaseelan K, Dayatilake GA, Diak R, Breen KC, Tustin DS, Costes E, Chagné D, Schaffer RJ, David KM (2012) A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control. BMC Plant Biol 12:7CrossRefPubMedPubMedCentralGoogle Scholar
  7. Filonova LH, Bozhkov PV, Brukhin VB, Daniel G, Zhivotovsky B, von Arnold S (2000) Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce. J Cell Sci 113:4399–4411PubMedGoogle Scholar
  8. Frigerio M, Alabadí D, Pérez-Gómez J, García-Cárcel L, Phillips AL, Hedden P, Blázquez MA (2006) Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis. Plant Physiol 142:553–563CrossRefPubMedPubMedCentralGoogle Scholar
  9. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb Viewer: An environment for comparative protein modeling. Electrophoresis 18:2714–2723CrossRefGoogle Scholar
  10. Hagen G, Guilfoyle TJ (1985) Rapid induction of selective transcription by auxins. Mol Cell Biol 5:1197–1203CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hagen G, Guilfoyle T (2002) Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol Biol 49:373–385CrossRefPubMedGoogle Scholar
  12. Jain M, Kaur N, Tyagi AK, Khurana JP (2006) The auxin-responsive GH3 gene family in rice (Oryza sativa). Funct Integr Genom 6:36CrossRefPubMedGoogle Scholar
  13. Jain M, Khurana JP (2009) Transcript profiling reveals diverse roles of auxin-responsive genes during reproductive development and abiotic stress in rice. FEBS J 276:3148–3162CrossRefPubMedPubMedCentralGoogle Scholar
  14. Larsson E (2011) Molecular regulation of embryo development in Norway spruce. Dissertation, Swedish University of Agricultural SciencesGoogle Scholar
  15. Li S-g, Li W-f, Han S-y, Yang W-h, Qi L-W (2013a) Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos. Gene 522:177–183CrossRefPubMedGoogle Scholar
  16. Li W-F, Zhang S-G, Han S-Y, Wu T, Zhang J-H, Qi L-W (2013b) Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr. Plant Cell Tissue Organ Cult 113:131–136CrossRefGoogle Scholar
  17. Li Z-X, Fan Y-R, Dang S-F, Li W-F, Qi L-W, Han S-Y (2018) LaMIR166a-mediated auxin biosynthesis and signalling affect somatic embryogenesis in Larix leptolepis. Mol Genet Genom 293:1355–1363CrossRefGoogle Scholar
  18. Li Z-X, Zhang L-F, Li W-F, Qi L-W, Han S-Y (2017) MIR166a affects the germination of somatic embryos in Larix leptolepis by modulating IAA biosynthesis and signaling genes. J Plant Growth Regul 36:889–896CrossRefGoogle Scholar
  19. Liu K, Kang B-C, Jiang H, Moore SL, Li H, Watkins CB, Setter TL, Jahn MM (2005) A GH3-like gene, CcGH3, isolated from Capsicum chinense L. fruit is regulated by auxin and ethylene. Plant Mol Biol 58:447–464CrossRefPubMedGoogle Scholar
  20. Liu Z-B, Ulmasov T, Shi X, Hagen G, Guilfoyle TJ (1994) Soybean GH3 promoter contains multiple auxin-inducible elements. Plant Cell 6:645–657PubMedPubMedCentralGoogle Scholar
  21. Möller B, Weijers D (2009) Auxin control of embryo patterning. Cold Spring Harb Perspect Biol 1:a001545CrossRefPubMedPubMedCentralGoogle Scholar
  22. Nakazawa M, Yabe N, Ichikawa T, Yamamoto YY, Yoshizumi T, Hasunuma K, Matsui M (2001) DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length. Plant J 25:213–221CrossRefPubMedGoogle Scholar
  23. Okrent RA, Wildermuth MC (2011) Evolutionary history of the GH3 family of acyl adenylases in rosids. Plant Mol Biol 76:489–505CrossRefPubMedGoogle Scholar
  24. Ostrowski M, Jakubowska A (2013) GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light, plant hormones and auxinic herbicides. J Plant Physiol 170:361–368CrossRefPubMedGoogle Scholar
  25. Park J-E, Park J-Y, Kim Y-S, Staswick PE, Jeon J, Yun J, Kim S-Y, Kim J, Lee Y-H, Park C-M (2007a) GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis. J Biol Chem 282:10036–10046CrossRefPubMedGoogle Scholar
  26. Park J-E, Seo PJ, Lee A-K, Jung J-H, Kim Y-S, Park C-M (2007b) An Arabidopsis GH3 gene, encoding an auxin-conjugating enzyme, mediates phytochrome B-regulated light signals in hypocotyl growth. Plant Cell Physiol 48:1236–1241CrossRefPubMedGoogle Scholar
  27. Reddy S, Hitchin S, Melayah D, Pandey AK, Raffier C, Henderson J, Marmeisse R, Gay G (2006) The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment. New Phytol 170:391–400CrossRefPubMedGoogle Scholar
  28. Round A, Brown E, Marcellin R, Kapp U, Westfall CS, Jez JM, Zubieta C (2013) Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography. Acta Crystallogr Sect D: Biol Crystallogr 69:2072–2080CrossRefGoogle Scholar
  29. Roux C, Perrot-Rechenmann C (1997) Isolation by differential display and characterization of a tobacco auxin-responsive cDNA Nt-gh3. related to GH3. FEBS Lett 419:131–136CrossRefPubMedGoogle Scholar
  30. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738CrossRefPubMedPubMedCentralGoogle Scholar
  31. Roy A, Yang J, Zhang Y (2012) COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Res 40:W471–W477CrossRefPubMedPubMedCentralGoogle Scholar
  32. Staswick PE, Serban B, Rowe M, Tiryaki I, Maldonado MT, Maldonado MC, Suza W (2005) Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17:616–627CrossRefPubMedPubMedCentralGoogle Scholar
  33. Staswick PE, Tiryaki I, Rowe ML (2002) Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation. Plant Cell 14:1405–1415CrossRefPubMedPubMedCentralGoogle Scholar
  34. Suza WP, Rowe ML, Hamberg M, Staswick PE (2010) A tomato enzyme synthesizes (+)-7-iso-jasmonoyl-L-isoleucine in wounded leaves. Planta 231:717–728CrossRefPubMedGoogle Scholar
  35. Takase T, Nakazawa M, Ishikawa A, Kawashima M, Ichikawa T, Takahashi N, Shimada H, Manabe K, Matsui M (2004) ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation. Plant J 37:471–483CrossRefPubMedGoogle Scholar
  36. Tanaka S-i, Mochizuki N, Nagatani A (2002) Expression of the AtGH3a gene, an Arabidopsis homologue of the soybean GH3 gene, is regulated by phytochrome B. Plant Cell Physiol 43:281–289CrossRefPubMedGoogle Scholar
  37. Terol J, Domingo C, Talón M (2006) The GH3 family in plants: genome wide analysis in rice and evolutionary history based on EST analysis. Gene 371:279–290CrossRefPubMedGoogle Scholar
  38. Vestman D (2012) Changes in global gene expression and auxin dynamics during embryo development in conifers. Dissertation, Swedish University of Agricultural SciencesGoogle Scholar
  39. von Arnold S, Larsson E, Moschou P, Zhu T, Uddenberg D, Bozhkov P (2016) Norway spruce as a model for studying regulation of somatic embryo development in conifers. Vegetative Propagation of Forest Trees, pp 351–372Google Scholar
  40. Wang S, Bai Y, Shen C, Wu Y, Zhang S, Jiang D, Guilfoyle TJ, Chen M, Qi Y (2010) Auxin-related gene families in abiotic stress response in Sorghum bicolor. Funct Integr Genom 10:533–546CrossRefPubMedGoogle Scholar
  41. Westfall CS, Herrmann J, Chen Q, Wang S, Jez JM (2010) Modulating plant hormones by enzyme action: the GH3 family of acyl acid amido synthetases. Plant Signal Behav 5:1607–1612CrossRefPubMedPubMedCentralGoogle Scholar
  42. Westfall CS, Sherp AM, Zubieta C, Alvarez S, Schraft E, Marcellin R, Ramirez L, Jez JM (2016) Arabidopsis thaliana GH3. 5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis. Proc Natl Acad Sci USA 113:13917–13922CrossRefPubMedGoogle Scholar
  43. Westfall CS, Zubieta C, Herrmann J, Kapp U, Nanao MH, Jez JM (2012) Structural basis for prereceptor modulation of plant hormones by GH3 proteins. Science 336:1708–1711CrossRefPubMedGoogle Scholar
  44. Yuan H, Zhao K, Lei H, Shen X, Liu Y, Liao X, Li T (2013) Genome-wide analysis of the GH3 family in apple (Malus × domestica). BMC Genom 14:297CrossRefGoogle Scholar
  45. Zhang C, Zhang L, Wang D, Ma H, Liu B, Shi Z, Ma X, Chen Y, Chen Q (2018) Evolutionary History of the Glycoside Hydrolase 3 (GH3) Family based on the sequenced genomes of 48 plants and identification of jasmonic acid-related GH3 proteins in Solanum tuberosum. Int J Mol Sci 19:1850CrossRefPubMedCentralGoogle Scholar
  46. Zhang J-H, Zhang S-G, Li S-G, Han S-Y, Li W-F, Li X-M, Qi L-W (2014a) Regulation of synchronism by abscisic-acid-responsive small noncoding RNAs during somatic embryogenesis in larch (Larix leptolepis). Plant Cell Tissue Organ Cult 116: 361–370CrossRefGoogle Scholar
  47. Zhang J, Zhang S, Han S, Li X, Tong Z, Qi L (2013a) Deciphering small noncoding rnas during thetransition from dormant embryo to germinated embryo in larches (Larix leptolepis). PLoS One 8(12):e81452CrossRefPubMedPubMedCentralGoogle Scholar
  48. Zhang J, Zhang S, Han S, Wu T, Li X, Li W, Qi L (2012a) Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta 236:647–657CrossRefPubMedGoogle Scholar
  49. Zhang L-f, Li W-f, Han S-y, Yang W-h, Qi L-w (2013b) cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis). Gene 529:150–158CrossRefPubMedGoogle Scholar
  50. Zhang L-f, Li W-f, Xu H-y, Qi L-w, Han S-y (2014b) Cloning and characterization of four differentially expressed cDNAs encoding NFYA homologs involved in responses to ABA during somatic embryogenesis in Japanese larch (Larix leptolepis). Plant Cell Tissue Organ Cult 117:293–304CrossRefGoogle Scholar
  51. Zhang S-W, Li C-H, Cao J, Zhang Y-C, Zhang S-Q, Xia Y-F, Sun D-Y, Sun Y (2009) Altered architecture and enhanced drought tolerance in rice via the down-regulation of indole-3-acetic acid by TLD1/OsGH3.13 activation. Plant Physiol 151:1889–1901CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinf 9:40CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zhang Y, Zhang S, Han S, Li X, Qi L (2012b) Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis). Plant Cell Rep 31:1637–1657CrossRefPubMedGoogle Scholar
  54. Zhang Z, Li Q, Li Z, Staswick PE, Wang M, Zhu Y, He Z (2007) Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction. Plant Physiol 145:450–464CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Tree Genetics and Breeding, Research Institute of ForestryChinese Academy of ForestryBeijingPeople’s Republic of China
  2. 2.Key Laboratory of Tree Breeding and Cultivation, Research Institute of ForestryNational Forestry and Grassland Administration, Chinese Academy of ForestryBeijingPeople’s Republic of China
  3. 3.Research Institute of Forestry Policy and InformationChinese Academy of ForestryBeijingChina
  4. 4.Research Institute of Forest Ecology, Environment and ProtectionChinese Academy of ForestryBeijingPeople’s Republic of China

Personalised recommendations