3 Biotech

, 9:15 | Cite as

Stochasticity in transcriptional expression of a negative regulator of Arabidopsis ABA network

  • Necla PehlivanEmail author
Original Article


Stably heritable spatiotemporal co/over-expression of distinct transcriptional regulators across generations is a desired target as they signal traffic in the cell. Here, the stability and expression pattern of AtHB7 (Arabidopsis homeodomain-leucine zipper class I) cDNA was characterized in 220 random population of transformed tomato clones where no AtHB7 orthologous has been identified in to date. Integration of p35S::AtHB7 casette was tested by the amplification of the stretches (700/425 bp) in the target by NPT II/AtHB7 oligos. Transcriptional expression pattern for the amplicons of the specific transcripts in the leaf tissues of transformants were determined by qRT-PCR. Transgene copy number was negatively correlated with transgene expression level, yet a majority of transformants (78%) carried single-copy of transgene. About 1:3 of the lines containing two-copy inserts showed less transcript expression. Heterologous CaMV 35S promoter drove AtHB7, illuminated no penalty on transgene expression levels, stability or plant phenotype under drought stress. Integration and expression analysis of transcription factors is of great significance for reliable prediction of gene dosing/functions in plant genomes so as to sustain breeding under abiotic stress to guarantee food security.


HDZip AtHB7 Tomato 35S Trans-gene expression stability Drought 



I, the corresponding author express my sincere thanks to Dr. Rina Iannacone for her precious help in this work.

Compliance with ethical standards

Conflict of interest

No conflict of interest does exist between third parties.


  1. Ashraf M (2010) Inducing drought tolerance in plants: recent advances. Biotechnol Adv 28:169–183CrossRefGoogle Scholar
  2. Brunner AM, Li J, DiFazio SP et al (2007) Genetic containment of forest plantations. Tree Genet Genomes 3:75–100CrossRefGoogle Scholar
  3. Causse M, Damidaux R, Rousselle P (2006) Traditional and enhanced breeding for quality traits in tomato, vol 2. In: Genetic improvement of solanaceous crops. CRC Press, FloridaGoogle Scholar
  4. Darnell CL, Tonner PD, Gulli JG, Schmidler SC, Schmid AK (2017) Systematic discovery of archaeal transcription factor functions in regulatory networks through quantitative phenotyping analysis. mSystems 19:1–20Google Scholar
  5. Ding Z, Fu L, Yan Y, Tie W, Xia Z, Wang W, Peng M, Hu W, Zhang J (2017) Genome-wide characterization and expression profiling of HD-Zip gene family related to abiotic stress in cassava. PLoS ONE 12:e0173043CrossRefGoogle Scholar
  6. English JJ, Jones JD (1998) Epigenetic instability and trans-silencing interactions associated with an SPT::Ac T-DNA locus in tobacco. Genetics 148:457–469PubMedPubMedCentralGoogle Scholar
  7. Halpin C (2005) Gene stacking in transgenic plants-the challenge for 21st century plant biotechnology. Plant Biotechnol 3:141–155CrossRefGoogle Scholar
  8. Hanson J, Regan S, Engstrom P (2002) The expression pattern of the homeobox gene ATHB13 reveals a conservation of transcriptional regulatory mechanisms between Arabidopsis and hybrid aspen. Plant Cell Rep 21:81–89CrossRefGoogle Scholar
  9. Hjellstrom M, Olsson ASB, Engstrom O, Soderman EM (2003) Constitutive expression of the water deficit-inducible homeobox gene ATHB7 in transgenic Arabidopsis causes a suppression of stem elongation growth. Plant Cell Environ 26:1127–1136CrossRefGoogle Scholar
  10. Hoffmann AA, Hercus MJ (2000) Environmental stress as an evolutionary force. Bioscience 50:217–226CrossRefGoogle Scholar
  11. Iannacone R, Mittempergher F, Morelli G, Panio G, Perito A, Ruberti I, Sessa G, Cellini F (2008) Influence of an Arabidopsis dominant negative athb2 mutant on tomato plant development. Acta Hort 789:263–276CrossRefGoogle Scholar
  12. Jeong JS, Kim YS, Redillas M et al (2013) OsNAC5 overexpression enlarges root diameter in rice plants leading to enhanced drought tolerance and increased grain yield in the field. Plant Biotechnol J 11:101–114CrossRefGoogle Scholar
  13. Kærn M, Elston TC, Blake WJ, Collins JJ (2005) Stochasticity in gene expression: from theories to phenotypes. Nat Rev Genet 6:451–464CrossRefGoogle Scholar
  14. Khuong TT, Crété P, Robaglia C, Caffarri S (2013) Optimisation of tomato micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number. Plant Cell Rep 32:1441–1454CrossRefGoogle Scholar
  15. Kosová K, Vítámvás P, Prášil IT, Renaut J (2011) Plant proteome changes under abiotic stress-contribution of proteomics studies to understanding plant stress response. J Proteom 74:1301–1322CrossRefGoogle Scholar
  16. Kuang Z, Ji Z, Boeke JD, Ji H (2018) Dynamic motif occupancy (DynaMO) analysis identifies transcription factors and their binding sites driving dynamic biological processes. Nucleic Acids Res 46:e2CrossRefGoogle Scholar
  17. Lechtenberg B, Schubert D, Forsbach A, Gils M, Schmidt R (2003) Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. Plant J 34:507:517CrossRefGoogle Scholar
  18. Leibbrandt NB, Snyman SJ (2003) Stability of gene expression and agronomic performance of a transgenic herbicide-resistant sugarcane line in South Africa. Crop Sci 43:671–677CrossRefGoogle Scholar
  19. Li J, Brunner AM, Meilan R, Strauss SH (2009) Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. Tree Physiol 29:299–312CrossRefGoogle Scholar
  20. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Hinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an SEREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10:1391–1406CrossRefGoogle Scholar
  21. Liu Q, Guo Q, Akbar S, Zhi Y et al (2017) Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy. Plant Biotechnol J 15:56–67CrossRefGoogle Scholar
  22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using a real-time quantitative PCR and the \({2^{--\Delta \Delta {C_{\text{T}}}}}\) method. Methods 25:402–408CrossRefGoogle Scholar
  23. Matzke MA, Mette MF, Matzke AJM (2000) Trans-gene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol 43:401–415CrossRefGoogle Scholar
  24. Meza TJ, Stangeland B, Mercy IS et al (2002) Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing. Nucleic Acids Res 30:4556–4566CrossRefGoogle Scholar
  25. Mittler R, Blumwald E (2010) Genetic engineering for modern agriculture: challenges and perspectives. Annu Rev Plant Biol 61:443–462CrossRefGoogle Scholar
  26. Morino K, Olsen OA, Shimamoto K (1999) Silencing of an aleurone-specific gene in transgenic rice is caused by a rearranged transgene. Plant J 17:275–285CrossRefGoogle Scholar
  27. Oh SJ, Kim YS, Kwon CW, Park HK, Jeong JS, Kim JK (2009) Overexpression of the transcription factor AP37 in rice improves grain yield under drought conditions. Plant Physiol 150:1368–1379CrossRefGoogle Scholar
  28. Olsson AS, Engström P, Soderman E (2004) The homeobox genes Athb12 and AtHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol 55:663–677CrossRefGoogle Scholar
  29. Peterson DG, Boehm KS, Stack SM (1997) Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), a plant containing high levels of polyphenolic compounds. Plant Mol Biol Rep 15:148–153CrossRefGoogle Scholar
  30. Phillips RL, Kaeppler SM, Olhoft P (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. Proc Natl Acad Sci 91:5222–5226CrossRefGoogle Scholar
  31. Prescott A, Martin C (1987) A rapid method for the quantitative assessment of levels of specific mRNAs in plants. Plant Mol Biol Rep 4:219–224CrossRefGoogle Scholar
  32. Ré DA, Capella M, Bonaventure G, Chan RL (2014) Arabidopsis AtHB7 and AtHB12 evolved divergently to fine tune processes associated with growth and responses to water stress. BMC Plant Biol 14:150CrossRefGoogle Scholar
  33. Schmidt R, Mieulet D, Hubbertene HM, Obata T, Hoefgen R, Fernie AR, Fisahn J, Segundo BS, Guiderdoni E, Jos HM et al (2013) SALT-RESPONSIVE ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice. Plant Cell 25:2115–2131CrossRefGoogle Scholar
  34. Schrick K, Nguyen D, Karlowski WM, Mayer KFX (2004) Start lipid/sterol binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors. Genome Biol 5:1–15CrossRefGoogle Scholar
  35. Takahashi MU, Nakagawa S (2017) Transcription factor genes in evolution of the human genome. The genome and genes series: evolutionary studies, vol 1. Springer, TokyoGoogle Scholar
  36. Vain P, James A, Worland B, Snape W (2002) Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor Appl Genet 105:878–889CrossRefGoogle Scholar
  37. Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 17:29–35CrossRefGoogle Scholar
  38. Warren AJ (2002) Eukaryotic transcription factors. Curr Opin Struct Biol 12:107–114CrossRefGoogle Scholar
  39. Xu RF, Li H, Qin RY et al (2015) Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep 5:11491CrossRefGoogle Scholar
  40. Xu W, Liu W, Ye R, Mazarei M et al (2018) A profilin gene promoter from switchgrass (Panicum virgatum L.) directs strong and specific transgene expression to vascular bundles in rice. Plant Cell Rep 37:587–597CrossRefGoogle Scholar
  41. Yamasaki T, Miyasaka H, Ohama T (2008) Unstable RNAi effects through epigenetic silencing of an inverted repeat transgene in Chlamydomonas reinhardtii. Genetics 180:1927–1944CrossRefGoogle Scholar
  42. Yilmaz A, Nishiyama MY, Fuentes BG, Souza GM, Janies D, Gray J, Grotewold E (2009) GRASSIUS: a platform for comparative regulatory genomics across the grasses. Plant Physiol 149:171–180CrossRefGoogle Scholar
  43. Zale J, Jung JH, Kim JY, Pathak B et al (2016) Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass. Plant Biotechnol J 14:661–669CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Biology DepartmentRecep Tayyip Erdogan UniversityRizeTurkey

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